US20110046060A1 - Coagulation factor IX compositions and methods of making and using same - Google Patents

Abstract

The present invention relates to compositions comprising factor IX coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of coagulation factor-related diseases, disorders, and conditions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• This application claims the priority benefit of U.S. Provisional Application Ser. Nos. 61/236,493 filed Aug. 24, 2009, 61/236,836, filed Aug. 25, 2009, 61/280,955, filed Nov. 10, 2009, 61/280,956 filed Nov. 10, 2009, and U.S. application Ser. No. 12/699,761 filed Feb. 3, 2010, all of which, and co-pending application under attorney docket number 32808-719.201 filed herewith, are incorporated herein by reference in their entirety for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
• This invention was made with government support under SBIR grant 2R44GM079873-02 awarded by the National Institutes of Health. The government has certain rights in the invention.
SEQUENCE LISTING
• The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 8, 2010, is named 32887262.txt, and is 1,578,514 bytes in size.
BACKGROUND OF THE INVENTION
• In hemophilia, the clotting of blood is disturbed by a lack of certain plasma blood clotting factors. Human factor IX (FIX) is a zymogen of a serine protease that is an important component of the intrinsic pathway of the blood coagulation cascade. In individuals who do not have FIX deficiency, the average half-life of FIX is short, approximately 18-24 hours. A deficiency of functional FIX, due to an X-linked disorder that occurs in about one in 30,000 males, results in hemophilia B, also known as Christmas disease, named after a young boy named Stephen Christmas who was found to be lacking this factor. Over 100 mutations of factor IX have been described; some cause no symptoms, but many lead to a significant bleeding disorder. When untreated, hemophilia B is associated with uncontrolled bleeding into muscles, joints, and body cavities following injury, and may result in death. Previously, treatments for the disease included administration of FIX prepared from human plasma derived from donor pools, which carried attendant risks of infection with blood-borne viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). More recently, recombinant FIX products have become commercially available.
• The in vivo activity of exogenously supplied factor IX is limited both by protein half-life and inhibitors of coagulation, including antithrombin III. Factor IX compositions typically have short half-lives, requiring frequent injections. Also, current FIX-based therapeutics requires intravenous administration due to poor bioavailability. Thus, there is a need for improved factor IX compositions with extended half-life and retention of activity when administered as part of a preventive and/or therapeutic regimen for hemophilia, including hemophilia B.
• Factor VII is a coagulation factor protein synthesized in the liver and secreted into the blood as a single chain zymogen with a molecular weight of approximately 50 kDa. The FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage, and the activated form, when complexed with tissue factor (TF), is able to convert both factor IX and factor X into their activated forms, leading to rapid thrombin generation and fibrin formation. Because the circulating half-life of rFVIIa is about 2.3 hours (“Summary Basis for Approval for NovoSeven©”, FDA reference number 96-0597), multiple and frequent administrations are required for the treatment of bleeding disorders in hemophiliacs and subjects with factor VII deficiency.
• Chemical modifications to a therapeutic protein can reduce its in vivo clearance rate and subsequent increase serum half-life. One example of a common modification is the addition of a polyethylene glycol (PEG) moiety, typically coupled to the protein via an aldehyde or N-hydroxysuccinimide (NHS) group on the PEG reacting with an amine group (e.g. lysine side chain or the N-terminus). However, the conjugation step can result in the formation of heterogeneous product mixtures that need to be separated, leading to significant product loss and complexity of manufacturing and does not result in a completely chemically-uniform product. Also, the pharmacologic function of the therapetuics protein may be hampered if amino acid side chains in the vicinity of its binding site are modified by the PEGylation process. Fusing an Fc domain to the therapeutic protein is another approach to increases the size of the therapeutic protein, hence reducing the rate of clearance through the kidney. Additionally, the Fc domain confers the ability to bind to, and be recycled from lysosomes by, the FcRn receptor, which results in increased pharmacokinetic half-life. Unfortunately, the Fc domain does not fold efficiently during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be solubilized and functional protein must be renatured from the misfolded aggregate. Such process is time-consuming, inefficient, and expensive. Accordingly, there remains a need for improved coagulation factor compositions with increased half-life which can be administered less frequently, and/or be produced by a simpler process at a cheaper cost.
SUMMARY OF THE INVENTION
• The present invention is directed to compositions and methods for the treatment or improvement of a condition or the enhancement of a parameter associated with the administration of coagulations factors IX and/or VII. In particular, the present invention provides compositions of fusion proteins comprising one or more extended recombinant polypeptides (XTEN). A subject XTEN is typically a non-repetitive sequence and unstructured conformation. XTEN is linked to a coagulation factor (“CF”) selected from factor IX (“FIX”), factor VII (“FVII”), factor VII-factor IX hybrids, and sequence variants thereof, resulting in a coagulation factor-XTEN fusion protein (“CFXTEN”). In part, the present disclosure is directed to pharmaceutical compositions comprising the fusion proteins and the uses thereof for treating coagulation factor-related diseases, disorders or conditions. The CFXTEN compositions have enhanced pharmacokinetic properties compared to CF not linked to XTEN, which may permit more convenient dosing and improved efficacy. In some embodiments, the CFXTEN compositions of the invention do not have a component selected the group consisting of: polyethylene glycol (PEG), albumin, antibody, and an antibody fragment.
• In some embodiments, the invention provides an isolated factor IX fusion protein, comprising a factor IX sequence that is at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identical to an amino acid sequence selected from Table 1. The factor IX having such sequence idendity is further linked to an extended recombinant polypeptide (XTEN) having at least about 100 to about 3000 amino acid residues. In one embodiment, the XTEN is linked to the C-terminus of the FIX or the FVII CF. In some embodiments, the invention provides an isolated factor VII fusion protein, comprising a factor VII that is at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identical to an amino acid sequence selected from Table 2. The factor VII having such sequence is linked to an extended recombinant polypeptide (XTEN).
• Non-limiting examples of CFXTEN with a single FIX or a single FVII linked to a single XTEN are presented in Table 41. In one embodiment, the invention provides a CFXTEN composition has at least about 80% sequence identity compared to a CFXTEN from Table 41, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 41. In some embodiments, the CF and the XTEN components of the fusion protein are linked via a cleavage sequence that is cleavable by a protease, including endogenous mammalian proteases. Examples of such protease include, but are not limited to, FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, thrombin, elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, TEV, enterokinase, rhinovirus 3C protease, and sortase A, or a sequence selected from Table 7. In one embodiment, a CFXTEN composition with a cleavage sequence has a sequence having at least about 80% sequence identity compared to a CFXTEN from Table 42, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 42. However, the invention also provides substitution of any of the CF sequences of Table 1 or Table 2 for a CF in a sequence of Table 42, and substitution of any XTEN sequence of Table 4 for an XTEN in a sequence of Table 42, and substitution of any cleavage sequence of Table 7 for a cleavage sequence in a sequence of Table 42. In CFXTEN embodiments having cleavage sequences, cleavage of the cleavage sequence by the protease releases the XTEN from the CF. In some embodiments of the foregoing, the CF component becomes biologically active or has an increase in activity upon its release from the XTEN by cleavage of the cleavage sequence, wherein the pro-coagulant activity is at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to the corresponding FIX or FVII not linked to XTEN.
• The invention provides isolated CFXTEN fusion proteins that comprise a second XTEN of about 36 to about 3000 amino acid residues, which can be identical or can be different from the first XTEN, wherein the second XTEN can be incorporated between any two adjacent domains of the CF, i.e., between the Gla, EFG1, EGF2, activating peptide and protease domains, or is incorporated within the sequence of an existing loop domain of a domain sequence of the CF, as described more fully in the Examples. In one embodiment, the first and the second XTEN can be an amino acid sequence selected from any one of Tables 4, or 9-13, or can exhibit at least at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence selected from Tables 4 and 9-13. In another embodiment, the isolated fusion protein comprises a second XTEN of about 36 to about 3000 amino acid residues. The fusion protein can adopt a multiple-XTEN configuration of Table 6, or a variation thereof.
• The invention provides CFXTEN compositions comprising XTEN linked to a factor VII comprising one or more heterologous cleavage sequences cleavable by the same or different pro-coagulant proteases. In some embodiments of the foregoing, the factor VII comprises a heterologous sequence of factor XI incorporated into or substituted for portions of the FVII sequence, resulting in factor VII-factor IX hybrid sequence variants. In some embodiments, a portion or the entirety of the sequence from the activation peptide domain of FIX is incorporated or substituted for FVII sequences bridging the region between the EFG2 and protease domains of the FVII component, resulting in compositions that can be activated as part of the intrinsic system of the coagulation cascade (e.g., activated factor XI). In such case, the factor VII-factor IX CFXTEN composition can be activated by a pro-coagulant protease in the absence of tissue factor, such that the CFXTEN can serve as a by-pass of factors VIII and IX in the intrinsic coagulation pathway when such factors are deficient (e.g., in hemophilia A or B) or when inhibitors to these factors are present. In one embodiment, the FVII-FIX sequence variant incorporates the full-length FIX AP domain plus at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12 or moreamino acids flanking adjacent amino acid residues on one or both sides of the R145-A146 and R180-V181 cleavage sites of the FIX AP domain (e.g., the sequence RVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE (SEQ ID NO: 1) in the case of 12 flanking amino acids on the N-terminus side and 5 flanking amino acids on the C-terminus side).
• In another embodiment, the CFXTEN FVII-FIX sequence variant comprises a heterologous FIX sequence exhibiting at least at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or 100% identity compared to the sequence: KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV (SEQ ID NO: 2), when optimally aligned.
• In other embodiments, the CFXTEN comprises FVII-FIX sequence variants that incorporate a portion of the FIX AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5, or more amino acids that flank one or both sides of the R145-A146 cleavage site (e.g., the sequence TSKLTRAETVFP (SEQ ID NO: 3) in the case of 6 flanking amino acids on either side of the cleavage site) or a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 or more amino acids that flank one or both sides of the R180-V181 cleavage site (e.g., the sequence and DFTRV (SEQ ID NO: 4) in the case of 4 amino acids on the N-terminal flank and valine as the C-terminus of the cleavage site from FIX). In one embodiment of the foregoing, the CFXTEN FVII-FIX sequence variant comprises a heterologous FIX sequence exhibiting at least at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or 100% identity compared to a sequence selected from TSKLTRAETVFP (SEQ ID NO: 3) and FNDFTRV (SEQ ID NO: 5), when optimally aligned.
• In another embodiment, the CFXTEN comprises a FVII-FIX sequence variant disclosed above that further includes the same AP cleavage sequence as a linker between the C-terminus of the FVII component and the XTEN component of the fusion protein, e.g., an N- to C-terminus configuration of FVII variant-AP sequence-XTEN, thereby permitting the release of the FVII variant component from the CFXTEN fusion protein when cleaved by the same pro-coagulant protease as per that of the FVII to FVIIa transition. In one embodiment, the FVII-FIX CFXTEN of any of the foregoing embodiments includes the factor XI cleavage sequence KLTRAET (SEQ ID NO: 6) as the linker between the FVII-FIX sequence and the XTEN, thereby permitting the release of the FVII variant component from the CFXTEN fusion protein by the initiation of the intrinsic coagulation cascade. In one embodiment, the invention provides a CFXTEN with a FVII-FIX hybrid sequence that exhibits at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity compared to a sequence from Table 43. In other embodiments, the invention provides a FVII-FIX sequence variant with incorporated FIX-derived AP cleavage sequence that is not linked to an XTEN. In one embodiment, the FVII-FIX sequence without an XTEN exhibits at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity as compared with a sequence from Table 43 without an XTEN.
• In one embodiment of the CFXTEN composition, the invention provides a fusion protein of formula I:
• 
  (XTEN)_x-CF-(XTEN)_y  I
• wherein independently for each occurrence, CF is a coagulation factor; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides a fusion protein of formula II:
• 
  (XTEN)_x-(CF)-(S)_y-(XTEN)_y  II
• wherein independently for each occurrence, CF is a coagulation factor a; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; x is either 0 or 1 and y is either 0 or 1 wherein x+y>1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein, wherein the fusion protein is of formula III:
• 
  (XTEN)_x-(S)_x-(CF)-(S)_y-(XTEN)_y  III
• wherein independently for each occurrence, CF is a coagulation factor; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula IV:
• 
  (Gla)-(XTEN)_u-(EGF1)-(XTEN)_y-(EGF2)-(XTEN)_w-(AP)-(XTEN)_x-(Pro)-(S)_y-(XTEN)_z  IV
• wherein independently for each occurrence, Gla is a Gla domain of FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP is an activator peptide of FIX; PRO is a protease domain of FIX; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+z≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula V:
• 
  (Gla)-(XTEN)_u(EGF1)-(XTEN)_v-(EGF2)-(AP1)-(XTEN)_w-(AP2)-(XTEN))_x-(Pro)-(S)_y-(XTEN)_z  V
• wherein independently for each occurrence, Gla is a Gla domain of FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP1 is the N-terminal sequence portion of the activator peptide domain of FIX that includes a first native cleavage sequence of the AP domain; AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes a second native cleavage sequence of the AP domain; PRO is a protease domain of FIX; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+z≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula VI:
• 
  (Gla)-(XTEN)_u-(EGF1)-(XTEN)_v-(EGF2)-(XTEN)_w-(Pro)-(S)_x—(XTEN)_y  VI
• wherein independently for each occurrence, Gla is a Gla domain of FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is a protease domain of FVII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1 with the proviso that u+v+w+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula VII:
• 
  (Gla)-(XTEN)_t-(EGF1)-(XTEN)_u-(EGF2)-(AP1)_v-(XTEN)_w(AP2)_x-(Pro)-(S)_y-(XTEN)_z  VII
• wherein independently for each occurrence, Gla is a Gla domain of FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is a protease domain of FVII; AP1 is the N-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence; AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t+u+w+z≧1; and XTEN is an extended recombinant polypeptide. In the embodiment, the CFXTEN composition can include the entirety of the FIX activator peptide domain sequence or one or both cleavage sequences from the activator peptide domain of factor IX, e.g., a sequence of at least about 3 to about 12 amino acids that flank the R145-A146 cleavage site and the sequence of at least about 1 to about 5 amino acids that flank the R180-V181 cleavage site, as described more fully above. The invention also contemplates substitution of any of the other cleavage sequences of Table 7 for the AP cleavage sequences.
• The CFXTEN compositions of the embodiments described herein can be evaluated for retention of activity (including after cleavage of any incorporated XTEN-releasing cleavage sites) using any appropriate in vitro assay disclosed herein (e.g., the assays of Table 40 or the assays described in the Examples), to determine the suitability of the configuration for use as a therapeutic agent in the treatment of a coagulation-factor related disease, disorder or condition. In one embodiment, the CFXTEN exhibits at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF not linked to XTEN. In another embodiment, the CF component released from the CFXTEN by enzymatic cleavage of the incorporated cleavage sequence linking the CF and XTEN components exhibits at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF not linked to XTEN.
• The XTEN of the CFXTEN compositions have at least about 200, or at least about 400, or at least about 800, or at least about 900, or at least about 1000, or at least about 2000, up to about 3000 amino acids residues. The XTEN of the CFXTEN fusion protein compositions is characterized in that they have one or more of the following characteristics: (a) at least a first XTEN comprises at least about 200 contiguous amino acids that exhibits at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity to a comparable length of an amino acid sequence selected from a sequence shown in Table 4; (b) the XTEN sequence lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithm prediction for epitopes within the XTEN sequence is based on a score of −5, or −6, or −7, or −8, or −9 or greater; (c) the XTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or even less; (d) the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN; (e) the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN; (f) the XTEN has greater than 90% random coil formation, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% random coil formation as determined by GOR algorithm; (g) the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by the Chou-Fasman algorithm; and (h) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the total amino acid residues of the XTEN.
• In another embodiment, the invention provides CFXTEN fusion proteins, wherein the XTEN is characterized in that the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN, the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN, the XTEN sequence has less than 5% amino acid residues with a positive charge, the XTEN sequence has greater than 90% random coil formation, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% random coil formation as determined by GOR algorithm; and the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by the Chou-Fasman algorithm. In some embodiments, no one type of amino acid constitutes more than 30% of the XTEN sequence of the CFXTEN.
• In another embodiment, the invention provides CFXTEN fusion proteins, wherein the XTEN is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the sequence motifs has about 9 to about 14 amino acid residues and wherein the sequence of any two contiguous amino acid residues does not occur more than twice in each of the sequence motifs consist of four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). In one embodiment, the XTEN is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the XTEN sequence consists of non-overlapping sequence motifs wherein the motifs are selected from Table 3.
• In some embodiments, the XTEN has a sequence in which no three contiguous amino acids are identical unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In other embodiment, the XTEN component of the CFXTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less. In the embodiments of this paragraph, the XTEN is characterized as “substantially non-repetitive.”
• In some embodiments, the invention provides CFXTEN comprising at least a second XTEN, wherein the XTEN sequence exhibits at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13.
• In some embodiments, CFXTEN fusion proteins exhibits enhanced pharmacokinetic properties compared to CF not linked to XTEN, wherein the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic window, increased time between consecutive doses results in blood concentrations within the therapeutic window, and decreased dose in moles over time that can be administered compared to a CF not linked to XTEN, yet still result in a blood concentration within the therapeutic window for that composition. In some embodiments, the terminal half-life of the CFXTEN fusion protein administered to a subject is increased at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold, or even higher as compared to CF not linked to XTEN and administered to a subject at a comparable dose. In other embodiments, the terminal half-life of the CFXTEN fusion protein administered to a subject is at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 21 days or greater. In other embodiments, the enhanced pharmacokinetic property is reflected by the fact that the blood concentrations that remain within the therapeutic window for the CFXTEN fusion protein for a given period are at least about two fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold longer, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold compared to CF not linked to XTEN and administered to a subject at a comparable dose. The increase in half-life and time spent within the therapeutic window permits less frequent dosing and decreased amounts of the fusion protein (in moles equivalent) that are administered to a subject, compared to the corresponding CF not linked to XTEN. In one embodiment, administration of a CFXTEN to a subject using a therapeutically-effective dose regimen results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least six-fold, or at least eight-fold, or at least 10-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold, or at least about 100-fold or higher between at least two consecutive C_maxpeaks and/or C_mintroughs for blood levels of the fusion protein compared to the corresponding CF not linked to the XTEN and administered using a comparable dose regimen to a subject.
• In some embodiments, the XTEN enhances thermostability of CF when linked to the XTEN wherein the thermostability is ascertained by measuring the retention of biological activity after exposure to a temperature of about 37° C. for at least about 7 days of the biologically active protein in comparison to the biologically active protein not linked to the XTEN. In one embodiment of the foregoing, the retention of biological activity increases by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or about 150%, at least about 200%, at least about 300%, or about 500% longer compared to the CF not linked to the XTEN.
• In some embodiments, the isolated CFXTEN fusion protein is configures to have reduced binding affinity for a clearance receptor as compared to the corresponding CF not linked to the XTEN. In one embodiment, the CFXTEN fusion protein exhibits binding affinity for a clearance receptor of the CF in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about 2% to about 10% of the binding affinity of the corresponding CF not linked to the XTEN. In another embodiment, a CFXTEN fusion protein with reduced affinity can have reduced active clearance and a corresponding increase in half-life of at least about 3-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 12-fold, or at least about 15-fold, or at least about 17-fold, or at least about 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer compared to the corresponding CF that is not linked to the XTEN.
• In some embodiments, the invention provides CFXTEN fusion proteins wherein the CFXTEN exhibits increased solubility of at least three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least 40-fold, or at least 60-fold at physiologic conditions compared to the CF not linked to XTEN.
• In some embodiments, CFXTEN fusion proteins exhibit an increased apparent molecular weight as determined by size exclusion chromatography, compared to the actual molecular weight. In some embodiments the CF comprising a FIX and at least a first XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800 kD, or at least about 2000 kD, while the actual molecular weight of each FIX component of the fusion protein is about 50 kD and the molecular weight of the fusion protein ranges from about 70 to about 125 kDa. In other embodiments, the CF comprising a FVII and at least a first XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800 kD, or at least about 2000 kD, while the actual molecular weight of each FIX component of the fusion protein is about 50 kD and the molecular weight of the fusion protein ranges from about 70 to about 125 kDa. Accordingly, the CFXTEN fusion proteins can have an apparent molecular weight that is about 6-fold greater, or about 8-fold greater, or about 10-fold greater, or about 12-fold greater, or about 15-fold greater than the actual molecular weight of the fusion protein. In some cases, the isolated CFXTEN fusion protein of any of the embodiments disclosed herein exhibit an apparent molecular weight factor under physiologic conditions that is greater than about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or greater than about 15.
• In some embodiments, administration of a therapeutically effective dose of a fusion protein of one of formulae I-VII to a subject in need thereof can result in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding CF not linked to the XTEN of and administered at a comparable dose to a subject. In other cases, administration of a therapeutically effective dose of a fusion protein of an embodiment of formulas I-VII to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective dose regimen of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to a CF not linked to XTEN and administered at a comparable dose.
• The fusion proteins of the disclosed compositions can be designed to have different configurations, N- to C-terminus, of a CF and XTEN and optional spacer sequences, including but not limited to XTEN-CF, CF-XTEN, XTEN-S-CF, CF-S-XTEN, XTEN-CF-XTEN, CF-CF-XTEN, XTEN-CF-CF, CF-S-CF-XTEN, XTEN-CF-S-CF, and multimers thereof. The choice of configuration can, as disclosed herein, confer particular pharmacokinetic, physico/chemical, or pharmacologic properties including, in the case of an incorporated cleavage sequence, the release of the CF with a concomitant increase in activity.
• In some embodiments, the CFXTEN fusion protein is characterized in that: (i) it has a longer half-life when administered to a subject compared to the corresponding coagulation factor not linked to the XTEN administered to a subject under an otherwise equivalent dose; (ii) when a smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor that lacks the XTEN administered to a subject under an otherwise equivalent dose regimen, the fusion protein achieves a comparable area under the curve (AUC) as the corresponding coagulation factor not linked to the XTEN; (iii) when a smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor that lacks the XTEN administered to a subject under an otherwise equivalent dose regimen, the fusion protein achieves a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; (iv) when the fusion protein is administered to a subject less frequently in comparison to the corresponding coagulation factor not linked to the XTEN administered to a subject using an otherwise equivalent molar amount, the fusion protein achieves a comparable area under the curve (AUC) as the corresponding coagulation factor not linked to the XTEN; (v) when the fusion protein is administered to a subject less frequently in comparison to the corresponding coagulation factor not linked to the XTEN administered to a subject using an otherwise equivalent molar amount, the fusion protein achieves a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; (vi) when an accumulatively smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor not linked to the XTEN administered to a subject under an otherwise equivalent dose period, the fusion protein achieves comparable area under the curve (AUC) as the corresponding coagulation factor not linked to the XTEN; or (vii) when an accumulatively smaller molar amount of the fusion protein is administered to a subject in comparison to the corresponding coagulation factor not linked to the XTEN administered to a subject under an otherwise equivalent dose period, the fusion protein achieves comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN.
• The invention provides a method of producing a fusion protein comprising a factor VII or factor IX or a factor VII-factor IX hybrid coagulation factor fused to one or more extended recombinant polypeptides (XTEN), comprising: (a) providing host cell comprising a recombinant polynucleotide molecule encoding the fusion protein (b) culturing the host cell under conditions permitting the expression of the fusion protein; and (c) recovering the fusion protein from the culture. In one embodiment of the method, the coagulation factor of the fusion protein has at least 90% sequence identity compared to a sequence selected from Table 1 or Table 2. In another embodiment of the method, the one or more XTEN of the expressed fusion protein has at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% sequence identity compared to a sequence selected from Table 4. In another embodiment of the method, the host cell is a eukaryotic cell. In another embodiment of the method, the host cell is CHO cell. In another embodiment of the method the isolated fusion protein is recovered from the host cell cytoplasm in substantially soluble form.
• The invention provides isolated nucleic acids comprising a polynucleotide sequence selected from (a) a polynucleotide encoding the fusion protein of any of the foregoing embodiments, or (b) the complement of the polynucleotide of (a). In one embodiment, the invention provides an isolated nucleic acid comprising a polynucleotide sequence that has at least 80% sequence identity, or about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% sequence identity compared to (a) a polynucleotide sequence of comparable length selected from Table 41 and Table 42; or (b) the complement of the polynucleotide of (a). The invention provides expression vectors comprising the nucleic acid of any of the embodiments hereinabove described in this paragraph. In one embodiment, the expression vector of the foregoing further comprises a recombinant regulatory sequence operably linked to the polynucleotide sequence. In another embodiment, the polynucleotide sequence of the expression vectors of the foregoing is fused in frame to a polynucleotide encoding a secretion signal sequence, which can be a CF native signal sequence. The invention provides a host cell that comprises an expression vector of any of the embodiments hereinabove described in this paragraph. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is a CHO cell. In another embodiment, the host cell is HEK cell.
• In one embodiment, the invention provides pharmaceutical compositions comprising the fusion protein of any of the foregoing embodiments and a pharmaceutically acceptable carrier. In another embodiment, the invention provides kits, comprising packaging material and at least a first container comprising the pharmaceutical composition of the foregoing embodiment and a label identifying the pharmaceutical composition and storage and handling conditions, and a sheet of instructions for the reconstitution and/or administration of the pharmaceutical compositions to a subject.
• The invention provides a method of treating a coagulopathy or a coagulation factor-related disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of a CFXTEN fusion protein of any of the foregoing embodiments. In one embodiment of the method, the coagulation-factor related condition is selected from bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand's disease), coagulopathies (any disorder of blood coagulation, including coagulation factor deficiencies), hemophilia B (aka Christmas disease), factor IX-related bleeding disorders, factor VII deficiency, hemophilia A, vascular injury, uncontrolled bleeding in subjects not suffering from hemophilia, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, and bleeding due to liver disease. In one embodiment of the method of treatment, the coagulopathy is hemophilia A. In one embodiment of the method of treatment, the coagulopathy is hemophilia B. In another embodiment of the method of treatment, the coagulopathy is factor VII deficiency. In another embodiment of the method of treatment, the CFXTEN is administered to a subject to control a bleeding episode. In another embodiment of the method of treatment, a CFXTEN comprising a factor VII-factor IX sequence hybrid is administered to a subject to control a bleeding episode, wherein the CFXTEN is activated by a pro-coagulant protease of the intrinsic coaguation cascade (e.g., activated factor XI). In another embodiment, the present invention provides a method of treating a clotting factor deficiency in a subject, comprising: administering to said subject a composition comprising a therapeutically effective amount of the factor VII provided herein.
• In some embodiments, the composition can be administered subcutaneously, intramuscularly, or intravenously. In one embodiment, the composition is administered at a therapeutically effective amount, wherein the administration results in a gain in time spent within a therapeutic window for the fusion protein compared to the corresponding CF of the fusion protein not linked to the XTEN and administered at a comparable dose to a subject. The gain in time spent within the therapeutic window can at least three-fold longer than the corresponding CF not linked to the XTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer than the corresponding CF not linked to XTEN. In some embodiments of the method of treatment, (i) a smaller molar amount of (e.g. of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 100 fold-less or greater) the fusion protein is administered in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; (ii) the fusion protein is administered less frequently (e.g., every two days, about every seven days, about every 14 days, about every 21 days, or about, monthly) in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; or (iii) an accumulative smaller molar amount (e.g. about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less) of the fusion protein is administered in comparison to the corresponding coagulation factor tnot linked to the XTEN under the otherwise same dose regimen the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN. The accumulative smaller molar amount is measured for a period of at least about one week, or about 14 days, or about 21 days, or about one month. In some embodiments of the method of treatment, the therapeutic effect is a measured parameter selected from blood concentrations of coagulation factor, prothrombin (PT) assay, activated partial prothrombin (aPTT) assay, bleeding time assay, whole blood clotting time (WBCT), and thrombelastography.
• In another embodiment, invention provides a method of treating a disease, disorder or condition, comprising administering the pharmaceutical composition described above to a subject using multiple consecutive doses of the pharmaceutical composition administered using a therapeutically effective dose regimen. In one embodiment of the foregoing, the therapeutically effective dose regimen can result in a gain in time of at least three-fold, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least about 30-fold, or at least about 50-fold, or at least about 100-fold longer time between at least two consecutive C_maxpeaks and/or C_mintroughs for blood levels of the fusion protein compared to the corresponding CF of the fusion protein not linked to the fusion protein and administered at a comparable dose regimen to a subject. In another embodiment of the foregoing, the administration of the fusion protein results in improvement in at least one measured parameter of a coagulation factor-related disease using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the fusion protein and administered to a subject d using a therapeutically effective regimen to a subject.
• The invention further provides use of the compositions comprising the fusion protein of any of the foregoing embodiments in the preparation of a medicament for treating a disease, disorder or condition in a subject in need thereof. In one embodiment of the foregoing, the disease, disorder or condition is selected from group consisting of bleeding disorders, coagulopathies, hemophilia B (aka Christmas disease), factor IX-related bleeding disorders, factor VII deficiency, vascular injury, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, and liver disease. Any of the disclosed embodiments can be practiced alone or in combination depending on the interested application.
INCORPORATION BY REFERENCE
• All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
• The features and advantages of the invention may be further explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments.
• FIG. 1 shows a schematic representation of exemplary CFXTEN (FIX-XTEN) fusion proteins.FIG. 1A shows the domain architecture of native FIX, with the gamma-carboxyglutamate domain, the EGF1 and EGF2 domains, the activation peptide, and the protease domain, with a linked XTEN at the C-terminus Arrows indicate the cleavage sites for the activation peptide domain.FIG. 1B shows a FIX molecule with an XTEN polypeptide attached to the C-terminus via a cleavage sequence, and indicates a site for proteolytic cleavage to release the XTEN (arrows indicate the cleavage sites for the activation peptide domain and the release point for the XTEN).
• FIG. 2 illustrates several examples of CXTEN configurations of FIX-XTEN and associated protease cleavage sites.FIG. 2A shows an FIX-XTEN with two proteolytic cleavage sites (arrows) within the activation peptide of FIX, and a C-terminus XTEN without a cleavage site linkage.FIG. 2B is similar to the configuration ofFIG. 2A, but the C-terminus XTEN is linked via a cleavage sequence, with the arrow indicating the release point.FIG. 2C shows three configurations of FIX-XTEN, with the XTEN integrated between the various domains of FIX.FIG. 2D shows an FIX-XTEN with the XTEN portion inserted into the activation peptide between the native cleavage sites, which would release the XTEN upon the proteolytic activation of FIX.FIG. 2E illustrates FIX-XTEN that contain multiple XTEN sequences inserted between different domains with the addition of a releasable XTEN at the C-terminus.FIG. 2F illustrates FIX-XTEN where the XTEN has been inserted within loop domains of FIX.
• FIG. 3 is a schematic of the coagulation cascade, showing both the extrinsic and intrinsic pathways.
• FIG. 4 shows several examples of CXTEN configurations of FVII-XTEN.FIG. 4A shows a FVII-XTEN that has not been activated.FIG. 4B shows a FVII-XTEN in which the peptide has been cleaved, resulting in an activated FVIIa-XTEN;FIG. 4C illustrates a FVII-XTEN composition with a cleavage sequence for releasable XTEN in which the FVII component has not been activated, containing a cleavage site for the activation protease (AP) and a second cleavage site for the release protease (RP).FIG. 4D shows a composition of activated FVIIa-XTEN containing a cleavage site for the release protease.
• FIG. 5 illustrates a strategy for FVII-XTEN design approach using internal XTEN.FIGS. 5A-D show exemplary sites for XTEN insertion between boundaries of the FVII domains with inactive FVII on the left and an activated form of FVII on the right (A: Insertion of XTEN between Gla and EGF1 domain, B: Insertion of XTEN between EGF1 and EGF2. C: Insertion of XTEN at C-terminus of activation peptide, D: Insertion of XTEN at N-terminus of activation peptide).FIG. 5E shows examples of FVII-XTEN in which the XTEN is located within external loops within individual domains fusion proteins, with inactive FVII on the left and FVIIa on the right. The activation peptide in FVII is shown as a thin line versus XTEN that is shown as a fat line.
• FIG. 6 illustrates essentially the same constructs asFIG. 5, but with an XTEN linked at the C-terminus of each construct.
• FIG. 7 is a schematic that shows some of the various locations in which XTEN can be inserted internal to the sequences of the coagulation factors FVII or FIX.
• FIG. 8 is a schematic of the key components of the clotting system.FIG. 7A: Normal clotting system with the intrinsic and extrinsic cascade components.FIG. 7 B illustrates a variation in which an inactive/low active form of FVII-XTEN (FVII*) is intended to bypass the FIX and FVIII components of the intrinsic system when activated endogenously after administration.
• FIG. 9 is a graph of the distribution of cell cluster size (gray bars) and FVII ELISA titers in ng/ml (black bars) by ELISA of clones from primary screening of pBC0014 CHO-K1 transformants (not all clones were labeled underneath the bars due to insufficient space)(see Example 25 for experimental details). Clones were sorted according to ELISA titer low to high (left to right).
• FIG. 10 is a graph of cell counts (white bars) and FVII titers in ng/ml (black bars) of the top pBC0014 clones (see Example 25 for experimental details). Clones were sorted according to ELISA titer, low to high (left to right).
• FIG. 11 is a graph of the ratio of FVII titer over cell count of the top pBC0014 clones (see Example 25 for experimental details). Clones were sorted according to the ratio, low to high (left to right).
• FIG. 12 is a Western blot of top pBC0014 clones according to ELISA, clotting, ELISA/cell count and clotting/cell count ratios (see Example 25 for experimental details). Clone 6G1 expressed a truncated product and was not evaluated further.
• FIG. 13 is a Western blot of the top pBC0016 clones according to ELISA, clotting, ELISA/cell count and clotting/cell count ratios (see Example 25 for experimental details).
• FIG. 14 is a Western blot of the top pBC0018 clones according to ELISA, clotting, ELISA/cell count and clotting/cell count ratios (see Example 25 for experimental details). Clone 3B2 expressed a truncated product and was not evaluated further.
• FIG. 15 shows purification of FVII-AE864 by anti-GLA affinity chromatography (see Example 26 for experimental details). SDS-PAGE analysis demonstrating the purification of FVII-AE864 from concentrated supernatant and the >90% purity of the EDTA eluted fractions.
• FIG. 16 shows activation of FVII-XTEN fusions to FVIIa-XTEN fusions by FXa treatment (see Example 26 for experimental details). SDS-PAGE analysis demonstrates the appearance of a light chain band under reducing conditions after FXa treatment, but not in the untreated sample. Additionally, there is a downwards shift in the upper band indicating the loss of the light chain.
• FIG. 17 shows an SDS-PAGE demonstrating auto-activation of FVII-XTEN fusions to FVIIa-XTEN fusions (see Example 26 for experimental details). SDS-PAGE analysis demonstrating appearance of a light chain band under reducing conditions after FXa treatment and after incubation at 4° C. at high concentration with CaCl₂. Additionally, there is a downwards shift in the upper band indicating the loss of the light chain.
• FIG. 18 shows SEC Analysis of FVII-AE864 and FVII-AE288 (see Example 26 for experimental details). The SEC shows a monodispersed population with minimal contamination and no aggregates at the void volume of the column (˜22 ml).
• FIG. 19 shows the purification of FVII-AE864 by anion exchange chromatography (see Example 26 for experimental details). The chromatograms depict the elution profiles of the total protein content and the FVII activity from a Macrocap Q column with the bulk of the activity eluting later than the contaminant proteins, creating a net 5-fold purification.
• FIG. 20 shows purification of FVII-AE864 by hydrophobic interaction chromatography (see Example 26 for experimental details). The chromatograms depict the elution profiles of the total protein content and the FVII activity from a toyopearl phenyl column with the bulk of the activity eluting earlier than the contaminant proteins, creating a net 2-fold purification
• FIG. 21 shows two chromatography outputs demonstrating removal of aggregated protein from monomeric FVII-AE864 with anion exchange chromatography (see Example 26 for experimental details).FIG. 21A is a chromatogram depicting the elution profile of FVII-XTEN from a macrocap Q column with two peaks eluting after the buffer related early peak.FIG. 21B shows SEC chromatograms of the early and late macrocap Q peaks demonstrating the absence of aggregates in the early peak.
• FIG. 22 shows results of ELISA or aPTT assays, showing FIX/cFXI/XTEN has enhanced activity compared to FIX-XTEN (see Example 29 for experimental details). Transiently expressed FIX constructs were assayed for antigen content by ELISA and for activity by aPTT based assays. While the antigen content of FIX-XTEN was similar to the FIX/cFXI/XTEN constructs the activity was significantly increased. This increase is attributed to the specific action of the FXI protease in the assays as the FIX/cTEV/XTEN does not show a significantly different activity to FIX-XTEN. Note the ELISA titer of the FIX sample is 197 ng/ml and is off the scale of the graph.
• FIG. 23 shows the pharmacokinetic profile after a single dose administered subcutaneously to rats, with the derived equivalent FVII concentration shown, as described in Example 30.
• FIG. 24 shows the pharmacokinetic profile after a single dose administered subcutaneously to rats, with the derived equivalent FIX concentration shown, as described in Example 31.
• FIG. 25 shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, administered either subcutaneously or intravenously, as described in Example 39. The compositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are presented as the plasma concentration versus time (h) after dosing and show, in particular, a considerable increase in half-life for the XTEN_AD836-GFP, the composition with the longest sequence length of XTEN. The construct with the shortest sequence length, the GFP-L288 had the shortest half-life.
• FIG. 26 shows an SDS-PAGE gel of samples from a stability study of the fusion protein of XTEN_AE864 fused to the N-terminus of GFP (see Example 40). The GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37° C. In addition, GFP-XTEN administered to cynomolgus monkeys was also assessed. Samples were withdrawn at 0, 1 and 7 days and analyzed by SDS PAGE followed by detection using Western analysis with antibodies against GFP.
• FIG. 27 shows three randomized libraries used for the third and fourth codons in the N-terminal sequences of clones from LCW546, LCW547 and LCW552 (see Example 14 for experimental details). The libraries were designed with the third and fourth residues modified such that all combinations of allowable XTEN codons were present at these positions, as shown. In order to include all the allowable XTEN codons for each library, nine pairs of oligonucleotides encoding 12 amino acids with codon diversities of third and fourth residues were designed, annealed and ligated into the NdeI/BsaI restriction enzyme digested stuffer vector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of the three libraries LCW0569 (SEQ ID NOS 773 and 774, respectively), LCW0570 (SEQ ID NOS 775 and 776, respectively), and LCW0571 (SEQ ID NOS 777 and 778, respectively).
• FIG. 28 shows a histogram of a retest of the top 75 clones after the optimization step, as described in Example 15, for GFP fluorescence signal, relative to the benchmark CBD_AM875 construct. The results indicated that several clones were now superior to the benchmark clones.
• FIG. 29 is a schematic of a combinatorial approach undertaken for the union of codon optimization preferences for two regions of the N-terminus 48 amino acids (see Example 16 for experimental details). The approach created novel 48mers at the N-terminus of the XTEN protein for evaluation of the optimization of expression that resulted in leader sequences that can be a solution for the expression of XTEN proteins where the XTEN is N-terminal to the CF.
• FIG. 30 shows an SDS-PAGE gel confirming the expression of preferred clones obtained from the XTEN N-terminal codon optimization experiments, in comparison to benchmark XTEN clones comprising CBD leader sequences at the N-terminus of the construct sequences, as described in Example 17.
• FIG. 31 is a schematic flowchart of representative steps in the assembly, production and the evaluation of a XTEN.
• FIG. 32 is a schematic flowchart of representative steps in the assembly of a CFXTEN polynucleotide construct encoding a fusion protein.Individual oligonucleotides501 are annealed intosequence motifs502 such as a 12 amino acid motif (“12-mer”), which is subsequently ligated with an oligo containing BbsI, andKpnI restriction sites503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of theXTEN gene504 is achieved. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes anoptional Flag sequence506 followed by a stopper sequence that is flanked by BsaI, BbsI, andKpnI sites507 and anFVII gene508, resulting in thegene500 encoding an XTEN-FVII fusion protein.
• FIG. 33 is a schematic flowchart of representative steps in the assembly of a gene encoding fusion protein comprising a CF and XTEN, its expression and recovery as a fusion protein, and its evaluation as a candidate CFXTEN product.
• FIG. 34 is a schematic representation of the design of CFXTEN expression vectors with different processing strategies.FIG. 34A shows an expression vector encoding XTEN fused to the 3′ end of the sequence encoding FVII. Note that no additional leader sequences are required in this vector.FIG. 7B depicts an expression vector encoding XTEN fused to the 5′ end of the sequence encoding FVII with a CBD leader sequence and a TEV protease site.FIG. 7C depicts an expression vector as inFIG. 7B where the CBD and TEV processing sites have been replaced with an optimized N-terminal leader sequence (NTS).FIG. 7D depicts an expression vector encoding an NTS sequence, an XTEN, a sequence encoding VFII, and than a second sequence encoding an XTEN.
• FIG. 35 shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured against protein standards of known molecular weight, with the graph output as absorbance versus retention volume, as described in Example 37. The glucagon-XTEN constructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results indicate an increase in apparent molecular weight with increasing length of XTEN moiety.
• FIG. 36 shows sequence alignments between portions of native FIX, native FVII, and FVII-FIX sequence hybrids (SEQ ID NOS 779-786, respectively, in order of appearance) with different portions of the AP domain incorporated in the portion of the molecule spanning the EGF2 and Pro domains. The legend provides construct names. Gaps in an individual sequence (dashes) represents stretches of non-homology to FIX but are otherwise continuous, linked sequences. The underlined amino acids are FIX-derived sequence.
DETAILED DESCRIPTION OF THE INVENTION
• Before the embodiments of the invention are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.
• Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.
DEFINITIONS
• As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
• As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
• The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
• As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
• The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
• The term “non-naturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
• The terms “hydrophilic” and “hydrophobic” refer to the degree of affinity that a substance has with water. A hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, T P, et al., Proc Natl Acad Sci USA (1981) 78:3824. Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
• A “fragment” is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity. A “variant” is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.
• As used herein, “internal XTEN” refers to XTEN sequences that have been inserted into the sequence of the coagulation factor. Internal XTENs can be constructed by insertion of an XTEN sequence into the sequence of a coagulation factor such as FIX or FVII, either by insertion between two adjacent amino acids or domains of the coagulation factor or wherein XTEN replaces a partial, internal sequence of the coagulation factor.
• As used herein, “terminal XTEN” refers to XTEN sequences that have been fused to or in the N- or C-terminus of the coagulation factor or to a proteolytic cleavage sequence at the N- or C-terminus of the coagulation factor. Terminal XTENs can be fused to the native termini of the coagulation factor. Alternatively, terminal XTENs can replace a terminal sequence of the coagulation factor.
• The term “XTEN release site” refers to a sequence in CFXTEN fusion proteins that can be recognized and cleaved by a mammalian protease, effecting release of an XTEN or a portion of an XTEN from the CFXTEN fusion protein. As used herein, “mammalian protease” means a protease that normally exists in the body fluids, cells or tissues of a mammal. XTEN release sites can be engineered to be cleaved by various mammalian proteases (a.k.a. “XTEN release proteases”) such as FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any protease that is present during a clotting event.
• “Activity” as applied to form(s) of a CFXTEN polypeptide provided herein, refers to retention of a biological activity of the native coagulation factor, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to either receptor or ligand binding, enzymatic activity, or an effect on coagulation generally known in the art for the coagulation factor.
• A “therapeutic effect” as applied to form(s) of a CFXTEN polypeptide provided herein, refers to a physiologic effect, including but not limited to the curing, mitigation, reversal, amelioration or prevention of disease or conditions in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals. A “therapeutically effective amount” means an amount of compound effective to prevent, alleviate, reverse or ameliorate symptoms of disease or a condition (e.g., a bleeding episode) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.
• “Isolated,” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”
• An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.
• A “chimeric” protein contains at least one fusion polypeptide comprising regions in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
• “Conjugated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components, by whatever means including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and in reading phase or in-frame. An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
• In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
• “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
• The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
• The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.
• “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.
• The terms “gene” and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
• “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences.
• “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
• The terms “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Generally, stringency of hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60° C. for long polynucleotides (e.g., greater than 50 nucleotides)—for example, “stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and three washes for 15 min each in 0.1×SSC/1% SDS at 60° C. to 65° C. Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSC concentration may be varied from about 0.1 to 2×SSC, with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nded., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.; specifically seevolume 2 and chapter 9. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
• The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• “Percent (%) sequence identity,” with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• The term “non-repetitiveness” as used herein in the context of a polypeptide refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence. The term “substantially non-repetitive” can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there isn't a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence. The term “repetitiveness” as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a “repetitive” sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non-repetitive sequences, rendering the full-length polypeptide substantially non-repetitive. The length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids. “Repetitiveness” used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
• A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
• “Serum degradation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37° C. The samples for these time points can be run on a Western blot assay and the protein is detected with an antibody. The antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred. In this exemplary method, the time point where 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serum degradation half-life or “serum half-life” of the protein.
• The term “t_1/2” as used herein means the terminal half-life calculated as ln(2)/K_el. K_elis the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes. The terms “t_1/2”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein.
• “Active clearance” means the mechanisms by which CF is removed from the circulation other than by filtration or coagulation, and which includes removal from the circulation mediated by cells, receptors, metabolism, or degradation of the CF.
• “Apparent molecular weight factor” and “apparent molecular weight” are related terms referring to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid sequence. The apparent molecular weight is determined using size exclusion chromatography (SEC) and similar methods compared to globular protein standards and is measured in “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel.
• The terms “hydrodynamic radius” or “Stokes radius” is the effective radius (Rh in nm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution's viscosity. In the embodiments of the invention, the hydrodynamic radius measurements of the XTEN fusion proteins correlate with the ‘apparent molecular weight factor’, which is a more intuitive measure. The “hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.
• “Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al. (1989). Physiologically relevant temperature ranges from about 25° C. to about 38° C., and preferably from about 35° C. to about 37° C.
• A “reactive group” is a chemical structure that can be coupled to a second reactive group. Examples for reactive groups are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Non-limiting examples for activation are the reaction of a carboxyl group with carbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxyl group into an azide function.
• “Controlled release agent”, “slow release agent”, “depot formulation” and “sustained release agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent. Different embodiments of the present invention may have different release rates, resulting in different therapeutic amounts.
• The terms “antigen”, “target antigen” and “immunogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody fragment or an antibody fragment-based therapeutic binds to or has specificity against.
• The term “payload” as used herein refers to a protein or peptide sequence that has biological or therapeutic activity; the counterpart to the pharmacophore of small molecules. Examples of payloads include, but are not limited to, cytokines, enzymes, hormones and blood and growth factors. Payloads can further comprise genetically fused or chemically conjugated moieties such as chemotherapeutic agents, antiviral compounds, toxins, or contrast agents. These conjugated moieties can be joined to the rest of the polypeptide via a linker that may be cleavable or non-cleavable.
• The term “antagonist”, as used herein, includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide. In the context of the present invention, antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of a biologically active protein.
• The term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
• “Activity” for the purposes herein refers to an action or effect of a component of a fusion protein consistent with that of the corresponding native biologically active protein, wherein “biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to receptor binding, antagonist activity, agonist activity, or a cellular or physiologic response.
• As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• A “therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial.
• The term “therapeutically effective dose regimen”, as used herein, refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.
I). General Techniques
• The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3^rdedition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds., 1987; the series “Methods in Enzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practical approach”, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman's The Pharmacological Basis of Therapeutics,” 11^thEdition, McGraw-Hill, 2005; and Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” 4^thedition, John Wiley & Sons, Somerset, N.J., 2000, the contents of which are incorporated in their entirety herein by reference.
II). Coagulation Factors
• The present invention relates in part to fusion protein compositions comprising coagulation factors (CF). As used herein, “coagulation factor” or “CF” refers to factor IX (FIX), factor VII (FVII), sequence combinations of FVII and FIX, or mimetics, sequence variants and truncated versions thereof.
(a) Factor IX
• “Factor IX” or “FIX” means a coagulation factor protein and species and sequence variants thereof, and includes, but is not limited to, the 461 single-chain amino acid sequence of human FIX precursor polypeptide (“prepro”) and the 415 single-chain amino acid sequence of mature human FIX. FIX includes any form of factor IX molecule with the typical characteristics of blood coagulation factor IX. As used herein “factor IX” and “FIX” are intended to encompass polypeptides that comprise the domains Gla (region containingy-carboxyglutamic acid residues), EGF1 and EGF2 (region containing sequences homologous to human epidermal growth factor), activation peptide (formed by residues R136-R180 of the mature FIX), and the C-terminal protease domain (“Pro”), or synonyms of these domains known in the art, or can be a truncated fragment or a sequence variant that retains at least a portion of the biological activity of the native protein. FIX or sequence variants have been cloned, as described in U.S. Pat. Nos. 4,770,999, 7,700,734, and cDNA coding for human factor IX has been isolated, characterized, and cloned into expression vectors (see, for example, Choo et al., Nature 299:178-180 (1982); Fair et al., Blood 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad. Sci., U.S.A. 79:6461-6464 (1982)).
• Human factor IX (FIX) is encoded by a single-copy gene residing on the X-chromosome at q27.1. The human FIX mRNA is composed of 205 bases for the 5′ untranslated region, 1383 bases for the prepro factor IX, a stop codon and 1392 bases for the 3′ untranslated region. The FIX polypeptide is 55 kDa, synthesized as a prepropolypetide chain composed of three regions: a signal peptide of 28 amino acids, a propeptide of 18 amino acids, which is required for gamma-carboxylation of glutamic acid residues, and a mature factor IX of 415 amino acids. The propeptide is an 18-amino acid residue sequence N-terminal to the gamma-carboxyglutamate domain. The propeptide binds vitamin K-dependent gamma carboxylase and then is cleaved from the precursor polypeptide of FIX by an endogenous protease, most likely PACE (paired basic amino acid cleaving enzyme), also known as furin or PCSK3. Without the gamma carboxylation, the Gla domain is unable to bind calcium to assume the correct conformation necessary to anchor the protein to negatively charged phospholipid surfaces, thereby rendering factor IX nonfunctional. Even if it is carboxylated, the Gla domain also depends on cleavage of the propeptide for proper function, since retained propeptide interferes with conformational changes of the Gla domain necessary for optimal binding to calcium and phospholipid. In humans, the resulting mature factor IX is secreted by liver cells into the blood stream as an inactive zymogen, a single chain protein of 415 amino acid residues that contains approximately 17% carbohydrate by weight (Schmidt, A. E., et al. (2003) Trends Cardiovasc Med, 13: 39). The mature factor IX is composed of several domains that in an N- to C-terminus configuration are: a Gla domain, an EGF1 domain, an EGF2 domain, an activation peptide (AP) domain, and a protease (or catalytic) domain. FIX contains two activation peptides formed by R145-A146 and R180-V181, respectively. Following activation, the single-chain FIX becomes a 2-chain molecule, in which the two chains are linked by a disulfide bond attaching the enzyme to the Gla domain. CFs can be engineered by replacing their activation peptides resulting in altered activation specificity. In mammals, mature FIX must be activated by activated factor XI to yield factor IXa. The protease domain provides, upon activation of FIX to FIXa, the catalytic activity of FIX. Activated factor VIII (FVIIIa) is the specific cofactor for the full expression of FIXa activity.
• Proteins involved in clotting include factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, Protein C, and tissue factor (“clotting proteins”). The majority of the clotting proteins is present in zymogen form that when activated exhibits a pro-coagulant protease activity to activate other clotting proteins, contributing to the intrinsic or extrinsic coagulation pathway and clot formation. In the intrinsic pathway of the coagulation cascade, FIX associates with a complex of activated factor VIII, factor X, calcium, and phospholipid. In the complex, FIX is activated by factor XIa. The activation of factor IX is achieved by a two-step removal of the activation peptide (Ala 146-Arg 180) from the molecule (Bajaj et al., Human factor IX and factor IXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is made at the Arg 145-Ala 146 site by either factor XIa or factor VIIa/tissue factor. The second and rate limiting cleavage is made at Arg 180-Val 181. The activation removes 35 residues. Activated human factor IX exists as a heterodimer of the C-terminal heavy chain (28 kDa) and an N-terminal light chain (18 kDa), which are held together by one disulfide bridge attaching the enzyme to the Gla domain. Factor IXa in turn activates factor X in concert with activated factor VIII. Alternatively, factors IX and X can both be activated by factor VIIa complexed with lipidated Tissue Factor, generated via the extrinsic pathway. Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, and thrombin in turn converts fibrinogen to fibrin to form the clot.
• Defects in the coagulation process can lead to bleeding disorders in which the time taken for clot formation is prolonged. Such defects can be congenital or acquired. For example, hemophilia A and B are inherited diseases characterized by deficiencies in factor VIII (FVIII) and FIX, respectively. Replacement therapy with these proteins, generally prepared as recombinant proteins, may be used in the therapeutic intervention of hemophilia B (Christmas Disease) and factor IX-related bleeding disorders. Factor IX can be used in the treatment of both conditions. In some cases, however, patients develop antibodies against the administered proteins that reduce or negate the efficacy of the treatment.
• The invention contemplates inclusion of FIX sequences in the CFXTEN compositions that have homology to FIX sequences, sequence fragments that are natural, such as from humans, non-human primates, mammals (including domestic animals), and non-natural sequence variants which retain at least a portion of the biologic activity or biological function of FIX and/or that are useful for preventing, treating, mediating, or ameliorating a coagulation factor-related disease, deficiency, disorder or condition (e.g., bleeding episodes related to trauma, surgery, of deficiency of a coagulation factor). Sequences with homology to human FIX can be found by standard homology searching techniques, such as NCBI BLAST.
• In one embodiment, the FIX incorporated into the subject compositions is a recombinant polypeptide with a sequence corresponding to a protein found in nature. In another embodiment, the FIX is a sequence variant, fragment, homolog, or a mimetics of a natural sequence that retains at least a portion of the biological activity of the corresponding native FIX. Table 1 provides a non-limiting list of amino acid sequences of FIX that are encompassed by the CFXTEN fusion proteins of the invention. Any of the FIX sequences or homologous derivatives to be incorporated into the fusion protein compositions can be constructed by shuffling individual mutations between the amino acid sequences of Table 1 and evaluated for activity. Those that retain at least a portion of the biological activity of the native FIX are useful for the fusion protein compositions of this invention. FIX that can be incorporated into a CFXTEN fusion protein includes a protein that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an amino acid sequence selected from Table 1.

• TABLE 1
  FIX amino acid and nucleic acid sequences

  	SEQ
  	ID
  Name	NO:	Amino AcidSequence
  FIX precursor
  	7	MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF
  polypeptide		VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK
  		DDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL
  		AENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQ
  		SFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG
  		VKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNS
  		YVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLR
  		STKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKG
  		KYGIYTKVSRYVNWIKEKTKLT
  FIXHomo
  	8	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  sapiens		PCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV
  		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  4	9	MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF
  from Patent		VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK
  US		DDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL
  20080214462		AENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQ
  		SFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG
  		VKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSY
  		VTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRS
  		TKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGK
  		YGIYTKVSRYVNWIKEKTKLT
  Sequence
  6	10	MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF
  from Patent		VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK
  US		DDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL
  20080214462		AENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQ
  		SFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG
  		VKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSY
  		VTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRS
  		TKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGK
  		YGIYTKVSRYVNWIKEKTKLT
  Sequence
  8	11	MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEF
  from Patent		VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK
  US		DDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEG
  20080214462		YRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQ
  		STQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVE
  		TGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVL
  		NSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDATCL
  		RSTKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEGCAM
  		KGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  2	12	MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFV
  from Patent		QGNLERECMEEKCSFEEPREVFENTEKITEFWKQYVDGDQCESNPCLNGGSCKD
  U.S. Pat. No. 7,125,841		DINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLA
  		ENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNPTEAETILDNITQGTQS
  		FNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGV
  		KITVVAGEHNIEETEHTEQKRNVIRAIIPHHNYNAAINKYNHDIALLELDEPLVLNS
  		YVTPICIADKEYTNIFLKFGSGYVSGWARVFHKGRSALVLQYLRVPLVDRATCLR
  		STKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKG
  		KYGIYTKVSRYVNWIKEKTKLT
  Sequence
  1	13	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  2	14	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  3	15	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  4	16	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  5	17	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  6	18	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
  Sequence
  8	19	YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESN
  from Patent		PCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV
  US		CSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI
  20080167219		LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVT
  		AAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLE
  		LDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVP
  		LVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIIS
  		WGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

(b) Factor VII.
• “Factor VII” or “FVII” means a coagulation factor protein and species and sequence variants thereof, and includes, but is not limited to, both the inactive and activated forms (unless indicated to the contrary) of the 406 single-chain amino acid sequence of human FVII, and the 444 amino acid sequence of the precursor protein. As used herein, factor VII and FVII encompass polypeptides that comprise the domains Gla (region containing Y-carboxyglutamic acid residues), EGF1 and EGF2 (region containing sequences homologous to human epidermal growth factor), an activation peptide domain that spans the sequence between the EGF2 and Pro domains, and a catalytic or peptidase S1 domain (“Pro” region containing the serine protease catalytic triad), or synonyms of these domains known in the art, or can be a truncated fragment or a sequence variant that retains at least a portion of the biological activity of the native protein. Factor VII (FVII), a vitamin K-dependent plasma protein produced by the liver, initially circulates in the blood as a zymogen. The main role of factor VII is to initiate the process of coagulation in conjunction with tissue factor (TF). Upon vessel injury, tissue factor is exposed to the blood and circulating factor VII. Once bound to TF, FVII is activated to become the activated form of factor VII (FVIIa) by different proteases, among which are thrombin (factor IIa), factor Xa, IXa, XIIa, and the FVIIa-TF complex itself. The FVII zymogen is activated by proteolytic cleavage at a single site, Arg¹⁵²-Ile¹⁵³, resulting in a two-chain protease linked by a single disulphide bond (FVIIa). FVIIa binds its cofactor, tissue factor (TF), to form a complex which can activate factor X (FX) to FXa, thereby initiating a coagulation cascade that results in fibrin formation and hemostasis. The complete nucleotide and amino acid sequences for human factor VII are known, and human FVII or sequence variants have been cloned, as described in U.S. Pat. Nos. 4,784,950, 5,833,982, 6,911,323, and 7,026,524.
• Current therapeutic uses of factor VII exist but can be problematic in the treatment of individuals exhibiting a deficiency in factor VII, factor VIII, or factor IX, and individuals with Von Willebrand's disease with FVIIa formulations. More specifically, individuals receiving factors VIII and IX in replacement therapy frequently develop antibodies to these proteins. Continuing treatment is exceedingly difficult because of the presence of these antibodies. Patients experiencing this problem are normally treated with an activated prothrombin complex known to consist of a mixture of active and inactive clotting enzymes, including factor VIIa. FVII also is utilized in connection with treatment of uncontrolled bleedings, such as trauma, and it is believed that factor VIIa is capable of activating factor X to factor Xa without binding to tissue factor, and this activation reaction is believed to occur primarily on activated blood platelets (Hedner et al. Blood Coagulation & Fibrinolysis, 2000; 11; 107-111).
• Sequence variants of factor VII, whether exhibiting substantially the same or better bioactivity than wild-type factor VII, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor VII, include, polypeptides having an amino acid sequence that differs from the sequence of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids. Such FVII variants are known in the art, including those described in U.S. Pat. Nos. 6,960,657, 7,176,288, 7,414,022, 7,700,733, 20060205036A1, 20080318276A1, and 20090011992A1, which are incorporated herein by reference.
• Recombinant FVIIa has been approved for the treatment of hemophilia A or B patients that have inhibitors to FVIII or FIX, and also is used to stop bleeding episodes or prevent bleeding associated with trauma and/or surgery. Recombinant FVIIa also has been approved for the treatment of patients with congenital FVII deficiency, and is increasingly being utilized in off-label uses, such as the treatment of bleeding associated with other congenital or acquired bleeding disorders, trauma, and surgery in hemophilic and non-hemophilic patients.
• The invention contemplates inclusion in the CFXTEN compositions sequences with homology to FVII sequences, sequence fragments, mimetics and non-natural sequence variants which retain at least a portion of the biologic activity or biological function of FVIIa that are useful for preventing, treating, mediating, or ameliorating a CF-related disease, deficiency, disorder or condition. In addition, beause of the comparatively long-half life of CFXTEN comprising FVII, compositions comprising the inactive form of FVII that can be activated by mammalian endogenous proteases (described more fully below) or undergo autoactivation represents a means to treat subjects with certain forms of chronic coagulopathies with what is essentially a “prodrug” form of FVII. Table 2 provides a list of sequences of FVII that are encompassed by the CFXTEN fusion proteins of the invention. FVII sequences or homologous derivatives constructed by shuffling individual mutations between species or families that retain at least a portion of the biological activity of the native CF are useful for the fusion proteins of this invention. FVII that can be incorporated into a CFXTEN fusion protein include a protein that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence selected from Table 2.

• TABLE 2
  Factor VII amino acid sequences

  	SEQ
  	ID
  Name	NO:	AminoAcid Sequence
  FVII
  	20	MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERE
  precursor		CKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICF
  polypeptide		CLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADG
  		VSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLC
  		GGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPST
  		YVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLL
  		DRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCK
  		GDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRS
  		EPRPGVLLRAPFP
  Human FVII	21	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQ
  (mature)		NGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKR
  		SCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECP
  		WQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG
  		DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  		FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYM
  		FCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  		VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	22	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKSLTRNGPLKVCPKGECPW
  		QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDE
  		QSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFV
  		RFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFC
  		AGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVS
  		QYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	23	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKSMTRVVGGKVCPKGECP
  		WQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG
  		DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  		FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYM
  		FCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  		VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	24	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKCGQRLRKSKVCPKGECP
  		WQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG
  		DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  		FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYM
  		FCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  		VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	25	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIKPRIVGGKVCPKGECPW
  		QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDE
  		QSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFV
  		RFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFC
  		AGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVS
  		QYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	26	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKKLTRAETVKVCPKGECP
  		WQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG
  		DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  		FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYM
  		FCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  		VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	27	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKDFTRVVGGKVCPKGECP
  		WQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG
  		DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  		FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYM
  		FCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  		VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant
  	28	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIQIRSVAKKVCPKGECPW
  		QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDE
  		QSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFV
  		RFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFC
  		AGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVS
  		QYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant	29	NAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQN
  		GGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRS
  		CRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPW
  		QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDE
  		QSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFV
  		RFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFC
  		AGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVS
  		QYIEWLQKLMRSEPRPGVLLRAPFP
  FVII variant
  	30	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQ
  		NGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKR
  		SCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKIEPRSPSQKVCPKGECPW
  		QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDE
  		QSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFV
  		RFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFC
  		AGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVS
  		QYIEWLQKLMRSEPRPGVLLRAPFP

III). Coagulation Factor Fusion Protein Compositions
• The present invention provides fusion protein compositions comprising coagulation factors (CF). One way to increase the circulation half-life of a therapeutic protein is to reduce the renal clearance of the protein. This may be achieved by conjugating the protein to a polymer that s capable of conferring an increased molecular size (or hydrodynamic radius) to the protein, and hence, reduces renal clearance. Thus, one object of the present invention is to provide improved FIX or FVII (or FVIIa) molecules with a longer circulation, or terminal half-life (thereby decreasing the number of necessary administrations) and that retain at least a portion of the activity of the native coagulation factors, thereby to treat coagulation deficiencies and uncontrolled bleedings more efficiently. In one aspect, the invention provides isolated monomeric fusion proteins of CF comprising the full-length sequence or sequence variants of a CF, such as FIX or FVII, covalently linked to extended recombinant polypeptides (“XTEN” or “XTENs”). As described more fully below, the fusion proteins optionally include spacer sequences that further comprise cleavage sequences to release the CF from the fusion protein when acted on by a protease.
• In one aspect, the invention provides an isolated fusion protein comprising at least a first biologically active coagulation factor protein covalently linked to one or more extended recombinant polypeptides (“XTEN”), resulting in a fusion protein composition (hereinafter “CFXTEN”). The term “CFXTEN”, as used herein, is meant to encompass fusion polypeptides that comprise one or more payload regions each comprising a biologically active CF that mediates one or more biological or therapeutic activities associated with a coagulation factor and at least one other region comprising at least a first XTEN polypeptide that serves as a carrier. In one embodiment, the coagulation factor is FIX or a sequence variant of FIX, as disclosed above (including sequences with homology to the sequences of Table 1). In another embodiment, the coagulation factor is FVII, which can include the activated form of FVII, or a sequence variant of FVII, as disclosed above (including sequences with homology with the sequences of Table 2). In the case of CFXTEN compositions of the invention comprising FVII, activation of the FVII component may be carried out by exposure to activated factor X, by auto-activation, or according to procedures known in the art, such as those disclosed by Osterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No. 4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively, factor VII can be activated by passing it through an ion-exchange chromatography column (see, e.g., Bjoern et al. Research Disclosure (1986) 269:564-565), such as Mono Q (Pharmacia fine Chemicals) or similar chromatography resins.
• The CF of the subject compositions, particularly those disclosed in Tables 1 and 2, together with their corresponding nucleic acid and amino acid sequences, are well known in the art and descriptions and sequences are available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent). Polynucleotide sequences may be a wild type polynucleotide sequence encoding a given CF (e.g., either full length or mature), or in some instances the sequence may be a variant of the wild type polynucleotide sequence (e.g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been optimized, for example, for expression in a particular species; or a polynucleotide encoding a variant of the wild type protein, such as a site directed mutant or an allelic variant. It is well within the ability of the skilled artisan to use a wild-type or consensus cDNA sequence or a codon-optimized variant of a CF to create CFXTEN constructs contemplated by the invention using methods known in the art and/or in conjunction with the guidance and methods provided herein, and described more fully in the Examples.
• The CF for inclusion in the CFXTEN of the invention include coagulation factors or sequence variants that are useful, when administered to a subject, for mediating or preventing or ameliorating a disease, disorder or condition associated with bleeding disorders, coagulation factor deficiencies or defects in a coagulation factor. Of particular interest are CFXTEN fusion protein compositions for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, or some other enhanced pharmaceutical property compared to native CF is sought, or for which increasing the terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and/or improve patient compliance. Thus, the CFXTEN fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of the bioactive CF by, for example, increasing the in vivo exposure or the length that the CFXTEN remains within the therapeutic window when administered to a subject, compared to a CF not linked to XTEN.
• In one embodiment, the CF incorporated into the subject compositions can be a recombinant polypeptide with a sequence corresponding to a protein found in nature. In another embodiment, the CF is a sequence variant, fragment, homolog, or mimetic of a natural sequence that retain at least a portion of the biological activity of the native CF. In non-limiting examples, a CF is a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1 or from Table 2. In one embodiment, a CFXTEN fusion protein comprises a single CF molecule linked to a single XTEN (e.g., an XTEN as described more fully below). In another embodiment, the CFXTEN comprises a first CF and a second molecule of the same CF, resulting in a fusion protein comprising the two CF linked to one or more XTEN in an N- to C-terminus configuration selected from Table 6. In another embodiment, the CFXTEN fusion protein comprises a single CF molecule linked to a first and a second XTEN, in which the CF is a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1 or from Table 2, and the first and/or the second XTEN are sequences that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a sequence selected from Table 4.
• The subject CFXTEN of the present invention exhibits an enhancement of one or more pharmacokinetic parameters compared to the native CF. The CFXTEN with enhanced pharmacokinetic parameters permits less frequent dosing or an enhanced pharmacologic effect, inlcuding but not limited to maintaining the biologically active CFXTEN within the therapeutic window between the minimum effective dose or blood concentration (C_min) and the maximum tolerated dose or blood concentration (C_max) for a longer period of time compared to the CF not linked to XTEN. In such cases, the linking of the CF to a fusion protein comprising a select XTEN sequence(s) can result in an improvement in these properties, making them more useful as therapeutic or preventive agents compared to CF not linked to XTEN. In some embodiments, the subject CFXTEN of the present invention has a cleavage sequence incorporated between the CF and the XTEN and the biologic activity of the CF component is enhanced by the release of the CF from the fusion protein by cleavage of the cleavage sequence by an endogenous protease, as described below.
IV). Xtended Recombinant Polypeptides
• In one aspect, the invention provides XTEN polypeptide compositions that are useful as a fusion protein partner to which CF is linked, resulting in a CFXTEN fusion protein. XTEN are generally extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions.
• XTENs have utility as a fusion protein partners in that they serve as a “carrier,” conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a CF protein to a create a fusion protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics of the compositions, amongst other properties described herein. Such fusion protein compositions have utility to treat certain coagulation factor-related diseases, disorders or conditions, as described herein. As used herein, “XTEN” specifically excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments).
• In some embodiments, the XTEN is a long polypeptide having greater than about 100 to about 3000 amino acid residues when used as a carrier or greater than 400 to about 3000 residues cumulatively when more than one XTEN unit is used in a single fusion protein. In other embodiments, when XTEN is used as a linker between fusion protein components or where an increase in half-life of the fusion protein is not needed but where an increase in solubility or some other physico/chemical property for the CF fusion partner component is desired, an XTEN sequence shorter than 100 amino acid residues, such as about 96, or about 84, or about 72, or about 60, or about 48, or about 36 amino acid residues are incorporated into a fusion protein composition with the CF to effect the property.
• The selection criteria for the XTEN to be linked to the biologically active proteins used to create the inventive fusion proteins compositions generally relate to attributes of physical/chemical properties and conformational structure of the XTEN that is, in turn, used to confer enhanced pharmaceutical and pharmacokinetic properties to the fusion proteins compositions. The XTEN of the present invention exhibits one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, and increased hydrodynamic (or Stokes) radii; properties that make them particularly useful as fusion protein partners. Non-limiting examples of the properties of the fusion proteins comprising CF that are enhanced by XTEN include increases in the overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate of absorption when administered subcutaneously or intramuscularly, and enhanced pharmacokinetic properties such as longer terminal half-life and increased area under the curve (AUC), slower absorption after subcutaneous or intramuscular injection (compared to CF not linked to XTEN and administered by a similar route) such that the C_maxis lower, which, in turn, results in reductions in adverse effects of the CF that, collectively, results in an increased period of time that a fusion protein of a CFXTEN composition administered to a subject retains therapeutic activity.
• A variety of methods and assays are known in the art for determining the physical/chemical properties of proteins such as the compositions comprising the inventive XTEN. Such properties include but are not limited to secondary or tertiary structure, solubility, protein aggregation, melting properties, contamination and water content. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.
• In one embodiment, XTEN is designed to behave like denatured peptide sequence under physiological conditions, despite the extended length of the polymer. “Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR. “Denatured conformation” and “unstructured conformation” are used synonymously herein. In some embodiments, the invention provides XTEN sequences that, under physiologic conditions, resemble denatured sequences that are largely devoid in secondary structure. In other cases, the XTEN sequences are substantially devoid of secondary structure under physiologic conditions. “Largely devoid,” as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the methods described herein.
• A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. In particular, secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974)Biochemistry,13: 222-45) and the Garnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat J F, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).
• In some embodiments, the XTEN sequences used in the subject fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In other cases, the XTEN sequences of the fusion protein compositions have a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In other cases, the XTEN sequences of the fusion protein compositions have a high degree of random coil percentage, as determined by the GOR algorithm. In some embodiments, an XTEN sequence have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR algorithm.
1. Non-Repetitive Sequences
• In some embodiments, XTEN sequences of the compositions are substantially non-repetitive. In general, repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers. These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystalline structures. In contrast, the low tendency of non-repetitive sequences to aggregate enables the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive. Typically, the CFXTEN fusion proteins comprise XTEN sequences of greater than about 100 to about 3000 amino acid residues wherein the sequences are substantially non-repetitive. In one embodiment, the XTEN sequences have greater than about 100 to about 3000 amino acid residues in which no three contiguous amino acids in the sequence are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In the foregoing embodiment, the XTEN sequence is “substantially non-repetitive.”
• The degree of repetitiveness of a polypeptide or a gene can be measured by computer programs or algorithms or by other means known in the art. Repetitiveness in a polypeptide sequence can, for example, be assessed by determining the number of times shorter sequences of a given length occur within the polypeptide. For example, a polypeptide of 200 amino acid residues has 192 overlapping 9-amino acid sequences (or 9-mer “frames”) and 198 3-mer frames, but the number of unique 9-mer or 3-mer sequences will depend on the amount of repetitiveness within the sequence. A score is generated (hereinafter “subsequence score”) that is reflective of the degree of repetitiveness of the subsequences in the overall polypeptide sequence. In the context of the present invention, “subsequence score” means the sum of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are presented in Example 44. In some embodiments, the present invention provides CFXTEN each comprising one or more XTEN in which the XTEN has a subsequence score less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5. In the embodiments hereinabove described in this paragraph, an XTEN with a subsequence score less than about 10 (i.e., 9, 8, 7, etc.) is “substantially non-repetitive.”
• The non-repetitive characteristic of XTEN imparts a CF fusion proteins a greater degree of solubility and less tendency to aggregate compared to polypeptides having repetitive sequences. These properties facilitate the formulation of XTEN-comprising pharmaceutical preparations containing extremely high drug concentrations, in some cases exceeding 100 mg/ml.
• Furthermore, the XTEN polypeptide sequences of the embodiments are designed to have a low degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity when administered to a mammal. Polypeptide sequences composed of short, repeated motifs largely limited to three amino acids, such as glycine, serine and glutamate, may result in relatively high antibody titers when administered to a mammal despite the absence of predicted T-cell epitopes in these sequences. This may be caused by the repetitive nature of polypeptides, as it has been shown that immunogens with repeated epitopes, including protein aggregates, cross-linked immunogens, and repetitive carbohydrates are highly immunogenic and can, for example, result in the cross-linking of B-cell receptors causing B-cell activation. (Johansson, J., et al. (2007) Vaccine, 25:1676-82; Yankai, Z., et al. (2006) Biochem Biophys Res Commun, 345:1365-71; Hsu, C. T., et al. (2000) Cancer Res, 60:3701-5); Bachmann M F, et al. Eur J. Immunol. (1995) 25(12):3445-3451).
2. Exemplary Sequence Motifs
• The present invention encompasses XTEN used as fusion partners that comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are non-repetitive. The non-repetitive criterion can be met despite the use of a “building block” approach using a library of sequence motifs that are multimerized to create the XTEN sequences. Thus, while an XTEN sequence may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered substantially non-repetitive.
• In one embodiment, XTEN have a non-repetitive sequence of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs, wherein each of the motifs has about 9 to 36 amino acid residues. In other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 14 amino acid residues. In still other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence component consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues. In these embodiments, it is preferred that the sequence motifs be composed mainly of small hydrophilic amino acids, such that the overall sequence has an unstructured, flexible characteristic. Examples of amino acids that are included in XTEN are, e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As a result of testing variables such as codon optimization, assembly polynucleotides encoding sequence motifs, expression of protein, charge distribution and solubility of expressed protein, and secondary and tertiary structure, it was discovered that XTEN compositions with enhanced characteristics mainly include glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues wherein the sequences are designed to be substantially non-repetitive. In one embodiment, XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is greater than about 100 to about 3000 amino acid residues, preferably greater than 400 to about 3000 residues in length. In some embodiments, XTEN have sequences of greater than about 100 to about 3000 amino acid residues wherein at least about 80% of the sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In yet other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%.
• In still other embodiments, XTENs comprise non-repetitive sequences of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non-overlapping sequence motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In yet other embodiments, XTENs consist of 12 amino acid sequence motifs wherein the amino acids are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif, and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In the foregoing embodiments hereinabove described in this paragraph, the XTEN sequences is substantially non-repetitive.
• In some embodiments, the invention provides compositions comprising non-repetitive XTEN sequence(s) of greater than about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of two or more non-overlapping sequence motifs selected from the amino acid sequences of Table 3. In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of two or more non-overlapping sequences selected from a single motif family of Table 3, resulting in a “family” sequence in which the overall sequence remains substantially non-repetitive. Accordingly, in these embodiments, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family of sequences of Table 3. In other embodiments, the XTEN comprises motif sequences from two or more of the motif families of Table 3.

• TABLE 3
  XTEN Sequence Motifs of 12 Amino Acids and
  Motif Families.

  	Motif Family*	SEQ ID NO:	MOTIF SEQUENCE
  	AD	31	GESPGGSSGSES
  	AD	32	GSEGSSGPGESS
  	AD	33	GSSESGSSEGGP
  	AD	34	GSGGEPSESGSS
  	AE, AM	35	GSPAGSPTSTEE
  	AE, AM, AQ	36	GSEPATSGSETP
  	AE, AM, AQ	37	GTSESATPESGP
  	AE, AM, AQ	38	GTSTEPSEGSAP
  	AF, AM	39	GSTSESPSGTAP
  	AF,AM	40	GTSTPESGSASP
  	AF, AM	41	GTSPSGESSTAP
  	AF, AM	42	GSTSSTAESPGP
  	AG, AM	43	GTPGSGTASSSP
  	AG,AM	44	GSSTPSGATGSP
  	AG, AM	45	GSSPSASTGTGP
  	AG, AM	46	GASPGTSSTGSP
  	AQ	47	GEPAGSPTSTSE
  	AQ
  	48	GTGEPSSTPASE
  	AQ	49	GSGPSTESAPTE
  	AQ
  	50	GSETPSGPSETA
  	AQ	51	GPSETSTSEPGA
  	AQ
  	52	GSPSEPTEGTSA
  	BC	53	GSGASEPTSTEP
  	BC	54	GSEPATSGTEPS
  	BC	55	GTSEPSTSEPGA
  	BC	56	GTSTEPSEPGSA
  	BD	57	GSTAGSETSTEA
  	BD	58	GSETATSGSETA
  	BD	59	GTSESATSESGA
  	BD
  	60	GTSTEASEGSAS
  	*Denotes individual motif sequences that, when used together in various permutations, results in a “family sequence”

• In other embodiments, the CFXTEN composition comprises a non-repetitive XTEN sequence of greater than about 100 to about 3000 amino acid residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping 36 amino acid sequence motifs selected from one or more of the polypeptide sequences of Tables 9-12.
• In those embodiments wherein the XTEN component of the CFXTEN fusion protein has less than 100% of its amino acids consisting of four to six amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consisting of the sequence motifs of Table 3, or less than 100% sequence identity compared with an XTEN from Table 3, the other amino acid residues are selected from any other of the 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence. In such cases where the XTEN component of the CFXTEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferred that the amino acids not be hydrophobic residues and should not substantially confer secondary structure of the XTEN component. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain few (e.g. less than 5%) or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation). Thus, in some embodiments, the XTEN component of the CFXTEN fusion protein comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) would have a sequence with less than 5% of the residues contributing to alpha-helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.
3. Length of Sequence
• In another aspect of the present invention, the invention encompasses CFXTEN compositions comprising carriers of XTEN polypeptides with extended length sequences. The present invention makes use of the discovery that increasing the length of the non-repetitive, unstructured polypeptides enhances the unstructured nature of the XTENs and correspondingly enhances the biological and pharmacokinetic properties of fusion proteins comprising the XTEN carrier. As described more fully in the Examples, proportional increases in the length of the XTEN, even if created by a fixed repeat order of single family sequence motifs (e.g., the four AE motifs of Table 3), result in a sequence with a higher percentage of random coil formation, as determined by GOR algorithm, compared to shorter XTEN lengths. In general, increasing the length of the unstructured polypeptide fusion partner, as described in the Examples, results in a fusion protein with a disproportionate increase in terminal half-life compared to fusion proteins with unstructured polypeptide partners with shorter sequence lengths.
• Non-limiting examples of XTEN contemplated for inclusion in the CFXTEN of the invention are presented in Table 4, below. In one embodiment, the invention provides CFXTEN compositions wherein the XTEN sequence length of the fusion protein(s) is greater than about 100 to about 3000 amino acid residues, and in some cases is greater than 400 to about 3000 amino acid residues, wherein the XTEN confers enhanced pharmacokinetic properties on the CFXTEN in comparison to CF not linked to XTEN. In some embodiments, the XTEN sequences of the CFXTEN compositions of the present invention can be about 100, or about 144, or about 288, or about 401, or about 500, or about 600, or about 700, or about 800, or about 900, or about 1000, or about 1500, or about 2000, or about 2500 or up to about 3000 amino acid residues in length. In other cases, the XTEN sequences can be about 100 to 150, about 150 to 250, about 250 to 400, 401 to about 500, about 500 to 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. In one embodiment, the CFXTEN can comprise an XTEN sequence wherein the sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a XTEN selected from Table 4. In some embodiments, the XTEN sequence is designed for optimized expression as the N-terminal component of the CFXTEN by inclusion of encoding nucleotides for an optimized N-terminal leader sequence (NTS) in the XTEN portion of the gene encoding the fusion protein. In one embodiment, the N-terminal XTEN sequence of the expressed CFXTEN has at least 90% sequence identity compared to the sequence of AE48 or AM48, AE624, or AE912 or AM923. In another embodiment, the XTEN has the N-terminal residues described in Examples 14-17.
• In other embodiments, the CFXTEN fusion protein comprises a first and a second XTEN sequence, wherein the cumulative total of the residues in the XTEN sequences is greater than about 400 to about 3000 amino acid residues and the XTEN can be identical or they can be different in sequence. In embodiments of the foregoing, the CFXTEN fusion protein comprises a first and a second XTEN sequence wherein the sequences each exhibit at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to at least a first or additionally a second XTEN selected from Table 4. Examples where more than one XTEN is used in a CFXTEN composition include, but are not limited to constructs with an XTEN linked to both the N- and C-termini of at least one CF.
• As described more fully below, the invention provides methods in which the CFXTEN is designed by selecting the length of the XTEN to confer a target half-life on a fusion protein administered to a subject. In general, XTEN lengths longer that about cumulative 400 residues incorporated into the CFXTEN compositions result in longer half-life compared to shorter cumulative lengths; e.g., shorter than about 280 residues. However, in another embodiment, CFXTEN fusion proteins are designed to comprise XTEN with a longer sequence length that is selected to additionally confer slower rates of systemic absorption after subcutaneous or intramuscular administration to a subject. In such embodiments, the C_maxis reduced in comparison to a comparable dose of a CF not linked to XTEN, thereby contributing to the ability to keep the CFXTEN within the therapeutic window for the composition. Thus, the XTEN confers the property of a depot to the administered CFXTEN, in addition to the other physical/chemical properties described herein.

• TABLE 4
  XTEN Polypeptides

  XTEN	SEQ ID
  Name	NO:	Amino Acid Sequence
  AE48	61	MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS
  AM48	62	MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
  AE144	63	GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
  		APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESAT
  		PESGPGSEPATSGSETPGTSTEPSEGSAP
  AF144	64	GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGT
  		APGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGES
  		STAPGTSPSGESSTAPGTSPSGESSTAP
  AE288	65	GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  		GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
  		PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
  		ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT
  		STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  AF504	66	GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
  		GSPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG
  		TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGAS
  		PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGP
  		GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
  		GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA
  		STGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
  		TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
  		GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP
  AF540	67	GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESP
  		GPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS
  		GTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSES
  		PSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST
  		PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGS
  		TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAP
  		GSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGT
  		APGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
  		SASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPE
  		SGSASPGSTSESPSGTAP
  AD576	68	GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSE
  		GGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESG
  		SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGES
  		PGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSS
  		GSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSES
  		GSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGG
  		SSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSS
  		ESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSES
  		GESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSES
  		GSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSS
  		GPGESS
  AE576	69	GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
  		APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
  		TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
  		PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
  		PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
  		GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
  		APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
  		GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
  		ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP
  		AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
  AF576	70	GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESP
  		GPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS
  		GTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSES
  		PSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTST
  		PESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGS
  		TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAP
  		GSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGT
  		APGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESG
  		SASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPE
  		SGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP
  AE624	71	MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPT
  		STEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
  		SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
  		ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
  		GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
  		ETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP
  		TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTE
  		PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP
  		AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
  		GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST
  		EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
  AD836	72	GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSG
  		SESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGG
  		SSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSS
  		ESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSS
  		GESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSE
  		GGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGG
  		SSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSG
  		GEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESS
  		GSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSES
  		GSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESG
  		SSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPS
  		ESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESP
  		GGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSG
  		SSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGS
  		ESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS
  AE864	73	GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
  		APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP
  		TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
  		PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
  		PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
  		GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
  		APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
  		GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
  		ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP
  		AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
  		GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  		GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT
  		PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES
  		ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT
  		STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  AF864	74	GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSA
  		SPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPS
  		GTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSG
  		ESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTS
  		ESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGT
  		SPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGP
  		GSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGT
  		APGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESP
  		SGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTP
  		ESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTS
  		TPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPG
  		TSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPG
  		PGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESS
  		TAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA
  		ESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTP
  		SGATGSP
  AG864	75	GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT
  		GSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG
  		TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGAS
  		PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP
  		GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
  		GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA
  		STGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSS
  		TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP
  		GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST
  		GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGT
  		SSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTP
  		GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGP
  		GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG
  		TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGT
  		SSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGAS
  		PGTSSTGSP
  AM875	76	GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA
  		SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSG
  		SETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEP
  		SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS
  		ESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
  		GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTAS
  		SSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
  		TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAG
  		SPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTP
  		GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGP
  		GSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSE
  		TPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPS
  		GTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPS
  		ASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS
  		STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSA
  		PGTSTEPSEGSAP
  AE912	77	MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPT
  		STEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
  		SEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
  		ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
  		GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS
  		ETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP
  		TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTE
  		PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP
  		AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
  		GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST
  		EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP
  		ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP
  		SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP
  		AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP
  		GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
  		APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  AM923	78	MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSE
  		GSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSES
  		PSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE
  		SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
  		TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG
  		PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
  		SAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPS
  		GATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSP
  		AGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEG
  		SPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSS
  		PGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSG
  		SETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPAT
  		SGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTST
  		EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPG
  		ASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATG
  		SPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE
  		GSAP
  AM1318	79	GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA
  		SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSG
  		SETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEP
  		SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS
  		ESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
  		GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTAS
  		SSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSP
  		TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSES
  		ATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT
  		SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAP
  		GTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGS
  		APGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESAT
  		PESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPS
  		GESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSS
  		PSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSP
  		GASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESST
  		APGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSG
  		ATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSES
  		ATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTS
  		TPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
  		TSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG
  		SPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSS
  		TGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
  BC 864	80	GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGT
  		EPSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATS
  		GTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTE
  		PSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTS
  		EPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSA
  		GSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGT
  		EPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATS
  		GTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGA
  		SEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGS
  		GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPS
  		GTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPG
  		SAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSE
  		PGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPA
  		TSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGS
  		GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA
  BD864	81	GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSG
  		SETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSET
  		ATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGA
  		GSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSG
  		SETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSET
  		ATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGA
  		GTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSG
  		SETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSE
  		SATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSAS
  		GSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSG
  		SETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSE
  		SATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSAS
  		GTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATS
  		ESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTST
  		EASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGA
  		GTSESATSESGAGSETATSGSETA
  Y288	82	GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEG
  		EGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSEGSEGEGSGEGSEGE
  		GGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGE
  		GSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGE
  		GSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGE
  		GSEGSGEGEGSEGSGE
  Y576	83	GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGE
  		GEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGE
  		GEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEG
  		EGSEGGSEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEGSEG
  		EGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEG
  		EGSEGSGEGEGGSEGSEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGE
  		GGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGE
  		GSEGSGEGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGSEGE
  		GSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEGSEGGSEGEGSEGGSEGEG
  		SEGSGEGEGSEGSGEGEGSGEGSEGEGGSEGGEGEGSEGGSEGEGSEGGSEGEG
  		GEGSGEGEGGGEGSEGEGSEGSGEGEGSGEGSE

4. XTEN Segments
• In one embodiment, the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues containing at least one polypeptide sequence segment selected from Tables 4, 9, 10, 11, 12, and 13 and wherein at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% or more of the remainder of the XTEN sequence contains hydrophilic amino acids and less than about 2% of the remainder of the XTEN consists of hydrophobic or aromatic amino acids or cysteine. In some embodiments, the XTEN contains multiple segments wherein the segments are identical or different. In another embodiment, the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues and comprises at least one sequence segment of at least about 100 to about 923, or at least about 100 to about 875, or at least about 100 to about 576, or at least about 100 to about 288, or at least about 100 to about 144 amino acid residues wherein the sequence segment(s) consists of at least three different types of amino acids and the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues in the sequence segment(s) constitutes at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid sequence of the sequence segment and at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% of the remainder of the XTEN sequence(s) consist of hydrophilic amino acids and less than about 2% of the remainder of the XTEN sequence(s) consists of hydrophobic or aromatic amino acids, or cysteine. In another embodiment, the invention provides an isolated CFXTEN fusion protein wherein the cumulative length of the XTEN component is greater than about 100 to about 3000 amino acid residues and comprises at least one sequence segment of at least about 200 to about 923, or at least about 200 to about 875, or at least about 200 to about 576, or at least about 200 to about 288 amino acid residues wherein the sequence segment(s) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues in the sequence segment(s) constitutes at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid sequence of the sequence segment and wherein the subsequence score of the segment is less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5, and at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% of the remainder of the XTEN sequence(s) consist of hydrophilic amino acids and less than about 2% of the remainder of the XTEN sequence(s) consists of hydrophobic, aromatic or cysteine amino acids.
5. N-Terminal XTEN Expression-Enhancing Sequences
• In some embodiments, the invention provides a short-length XTEN sequence incorporated as the N-terminal portion of the CFXTEN fusion protein. It has been discovered that the expression of the fusion protein is enhanced in a host cell transformed with a suitable expression vector comprising an optimized N-terminal leader polynucleotide sequence (that encodes the N-terminal XTEN) incorporated into the polynucleotide encoding the binding fusion protein. As described in Examples 14-17, a host cell transformed with such an expression vector comprising an optimized N-terminal leader sequence (NTS) in the binding fusion protein gene results in greatly-enhanced expression of the fusion protein compared to the expression of a corresponding fusion protein from a polynucleotide not comprising the NTS, and obviates the need for incorporation of a non-XTEN leader sequence used to enhance expression. In one embodiment, the invention provides CFXTEN fusion proteins comprising an NTS wherein the expression of the binding fusion protein from the encoding gene in a host cell is enhanced about 50%, or about 75%, or about 100%, or about 150%, or about 200%, or about 400% compared to expression of a CFXTEN fusion protein not comprising the N-terminal XTEN sequence (where the encoding gene lacks the NTS).
• In one embodiment, the N-terminal XTEN polypeptide of the CFXTEN comprises a sequence that exhibits at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least 99%, or exhibits 100% sequence identity compared to the amino acid sequence of AE48 or AM48, the respective amino acid sequences of which are as follows:

• AE48:

  (SEQ ID NO: 61)

  MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS

  AM48:

  (SEQ ID NO: 62)

  MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS

• In another embodiment, the short-length N-terminal XTEN is linked to an XTEN of longer length to form the N-terminal region of the CFXTEN fusion protein, wherein the polynucleotide sequence encoding the short-length N-terminal XTEN confers the property of enhanced expression in the host cell, and wherein the long length of the expressed XTEN contributes to the enhanced properties of the XTEN carrier in the fusion protein, as described above. In the foregoing, the short-length XTEN is linked to any of the XTEN disclosed herein (e.g., an XTEN of Table 3) and the resulting XTEN, in turn, is linked to the N-terminal of any of the CF disclosed herein (e.g., a CF of Table 1 or Table 2) as a component of the fusion protein. Alternatively, polynucleotides encoding the short-length XTEN (or its complement) is linked to polynucleotides encoding any of the XTEN (or its complement) disclosed herein and the resulting gene encoding the N-terminal XTEN, in turn, is linked to the 5′ end of polynucleotides encoding any of the CF (or to the 3′ end of its complement) disclosed herein. In some embodiments, the N-terminal XTEN polypeptide with long length exhibits at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least 99%, or exhibits 100% sequence identity compared to an amino acid sequence selected from the group consisting of the sequences AE624, AE912, and AM923.
• In any of the foregoing N-terminal XTEN embodiments described above, the N-terminal XTEN can have from about one to about six additional amino acid residues, preferably selected from GESTPA, to accommodate the restriction endonuclease restriction sites that is employed to join the nucleotides encoding the N-terminal XTEN to the gene encoding the targeting moiety of the fusion protein. The methods for the generation of the N-terminal sequences and incorporation into the fusion proteins of the invention are described more fully in the Examples.
6. Net Charge
• In other embodiments, the XTEN polypeptides have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and/or reducing the proportion of hydrophobic amino acids in the XTEN sequence. The overall net charge and net charge density is controlled by modifying the content of charged amino acids in the XTEN sequences. In some embodiments, the net charge density of the XTEN of the compositions may be above +0.1 or below −0.1 charges/residue. By “net charge density” of a protein or peptide herein is meant the net charge divided by the total number of amino acids in the protein or proptide. In other embodiments, the net charge density of a XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.
• Since most tissues and surfaces in a human or animal have a net negative charge, in some embodiments, the XTEN sequences are designed to have a net negative charge to minimize non-specific interactions between the XTEN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that individually carry a net negative charge and that are distributed across the sequence of the XTEN polypeptide. Such a distribution of net negative charge in the extended sequence lengths of XTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. In preferred embodiments, the negative charge is conferred by incorporation of glutamic acid residues. Accordingly, in one embodiment the invention provides XTEN in which the XTEN sequences contain about 8, 10, 15, 20, 25, or even about 30% glutamic acid. Generally, the glutamic residues is spaced uniformly across the XTEN sequence. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20 kDa of XTEN that can result in an XTEN with charged residues that would have very similar pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the physicochemical properties of the resulting CFXTEN fusion protein for, and hence, simplifying purification procedures.
• The XTEN of the compositions of the present invention generally have no or a low content of positively charged amino acids. In some embodiments, the XTEN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% amino acid residues with a positive charge. However, the invention contemplates constructs where a limited number of amino acids with a positive charge, such as lysine, are incorporated into XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the XTEN backbone. In one embodiment of the foregoing, the XTEN has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or alternatively only a single lysine residue. Using the foregoing lysine-containing XTEN, fusion proteins are constructed that comprises XTEN, a coagulation factor, plus a chemotherapeutic agent useful in the treatment of growth-related diseases or disorders, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of lysines or other amino acids with reactive side chains (e.g., cysteine) incorporated into the XTEN.
• In some embodiments, the XTEN sequence comprises charged residues separated by other residues such as serine or glycine, which leads to better expression or purification behavior. Based on the net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 4.5. In these embodiments, the XTEN incorporated into the CFXTEN fusion protein compositions of the present invention carry a net negative charge under physiologic conditions that contribute to the unstructured conformation and reduced binding of the XTEN component to mammalian proteins and tissues.
• As hydrophobic amino acids impart structure to a polypeptide, the invention provides that the content of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In one embodiment, the amino acid content of methionine and tryptophan in the XTEN component of a CFXTEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%. In another embodiment, the XTEN will have a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total XTEN sequence.
7. Low Immunogenicity
• In another aspect, the invention provides compositions in which the XTEN sequences have a low degree of immunogenicity or are substantially non-immunogenic. Several factors can contribute to the low immunogenicity of XTEN, e.g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.
• Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The precise folding of the protein brings these sequences into a well-defined, stable spatial configurations, or epitopes, that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein or the activation of a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individual's HLA-DR allotype. Engagement of a MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
• The ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) is dependent on a number of factors; most notably its primary sequence. In one embodiment, a lower degree of immunogenicity is achieved by designing XTEN sequences that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well. The invention provides CFXTEN fusion proteins with substantially non-repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoiding formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Avoidance of immunogenicity can attribute to, at least in part, a result of the conformational flexibility of XTEN sequences; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The administration of fusion proteins comprising XTEN, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the CF fusion partner in the CFXTEN compositions.
• In one embodiment, the XTEN sequences utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003)J Immunol Methods,281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This is achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the XTEN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the XTEN sequences are substantially non-immunogenic by the restriction of the numbers of epitopes of the XTEN predicted to bind MHC receptors. With a reduction in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown in Example 45. The TEPITOPE score of a given peptide frame within a protein is the log of the K_d(dissociation constant, affinity, off-rate) of the binding of that peptide frame to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999)Nature Biotechnology17:555). The score ranges over at least 20 logs, from about 10 to about −10 (corresponding to binding constraints of 10e¹⁰K_dto 10e⁻¹⁰K_d), and can be reduced by avoiding hydrophobic amino acids that serve as anchor residues during peptide display on MHC, such as M, I, L, V, F. In some embodiments, an XTEN component incorporated into a CFXTEN does not have a predicted T-cell epitope at a TEPITOPE score of about −5 or greater, or −6 or greater, or −7 or greater, or −8 or greater, or at a TEPITOPE score of −9 or greater. As used herein, a score of “−9 or greater” would encompass TEPITOPE scores of 10 to −9, inclusive, but would not encompass a score of −10, as −10 is less than −9.
• In another embodiment, the inventive XTEN sequences, including those incorporated into the subject CFXTEN fusion proteins, are rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the XTEN, reducing the processing of XTEN into small peptides that can bind to MHC II receptors. In another embodiment, the XTEN sequence is rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the XTEN render the XTEN compositions, including the XTEN of the CFXTEN fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system. In one embodiment, an XTEN of a CFXTEN fusion protein can have >100 nM K_dbinding to a mammalian receptor, or greater than 500 nM K_d, or greater than 1 μM K_dtowards a mammalian cell surface or circulating polypeptide receptor.
• Additionally, the non-repetitive sequence and corresponding lack of epitopes of XTEN limit the ability of B cells to bind to or be activated by XTEN. A repetitive sequence is recognized and can form multivalent contacts with even a few B cells and, as a consequence of the cross-linking of multiple T-cell independent receptors, can stimulate B cell proliferation and antibody production. In contrast, while a XTEN can make contacts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual XTEN due to the lack of repetitiveness of the sequence. Not being to be bound by any theory, XTENs typically have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In one embodiment, the CFXTEN have reduced immunogenicity as compared to the corresponding CF that is not fused to an XTENT. In one embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-CF IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-XTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.
• An additional feature of XTENs with non-repetitive sequences relative to sequences with a high degree of repetitiveness is non-repetitive XTENs form weaker contacts with antibodies. Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and IgMs contain 10 identical binding sites. Thus antibodies against repetitive sequences can form multivalent contacts with such repetitive sequences with high avidity, which can affect the potency and/or elimination of such repetitive sequences. In contrast, antibodies against non-repetitive XTENs may yield monovalent interactions, resulting in less likelihood of immune clearance such that the CFXTEN compositions can remain in circulation for an increased period of time.
8. Increased Hydrodynamic Radius
• In another aspect, the present invention provides XTEN in which the XTEN polypeptides have a high hydrodynamic radius that confers a corresponding increased apparent molecular weight to the CFXTEN fusion protein incorporating the XTEN. As detailed in Example 38, the linking of XTEN to CF sequences, such as FIX or FVII sequences, results in CFXTEN compositions that can have increased hydrodynamic radii, increased apparent molecular weight, and increased apparent molecular weight factor compared to a CF not linked to an XTEN. For example, in therapeutic applications in which prolonged half-life is desired, compositions in which a XTEN with a high hydrodynamic radius is incorporated into a fusion protein comprising CF can effectively enlarge the hydrodynamic radius of the composition beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDA, which is larger than both native FIX and FVII) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced renal clearance of circulating proteins. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape or compactness. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. The open, extended and unstructured conformation of the XTEN polypeptide can have a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. As the results of Example 38 demonstrate, the addition of increasing lengths of XTEN results in proportional increases in the parameters of hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, permitting the tailoring of CFXTEN to desired characteristic cut-off apparent molecular weights or hydrodynamic radii. Accordingly, in certain embodiments, the CFXTEN fusion protein can be configured with an XTEN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In the foregoing embodiments, the large hydrodynamic radius conferred by the XTEN in an CFXTEN fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and/or a decrease in renal clearance rate.
• In another embodiment, an XTEN of a chosen length and sequence (e.g., a sequence from Table 4 or a sequence variant thereof) can be selectively incorporated into a CFXTEN to create a fusion protein that have, under physiologic conditions, an apparent molecular weight of at least about 500 kDa, or at least about 800 kDa, or at least about 1000 kDa, or at least about 1500 kDA, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In another embodiment, an XTEN of a chosen length and sequence can be selectively linked to a CF to result in a CFXTEN fusion protein that has, under physiologic conditions, an apparent molecular weight factor of at least four, alternatively of at least five, alternatively of at least six, alternatively of at least eight, alternatively of at least 10, alternatively of at least 15, or an apparent molecular weight factor of at least 20 or greater. In another embodiment, the CFXTEN fusion protein has, under physiologic conditions, an apparent molecular weight factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein.
V). CFXTEN Variants, Structural Configurations and Properties
• The CF of the subject compositions are not limited to native, full-length FIX or FVII polypeptides, but also include recombinant versions as well as biologically and/or pharmacologically active forms with sequence variants, combinations of FVII and FIX sequences, or fragments thereof. For example, it will be appreciated that various amino acid deletions, insertions and substitutions can be made in the CF to create variants without departing from the spirit of the invention with respect to the biological activity or pharmacologic properties of the CF. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 5. However, in embodiments of the CFXTEN in which the sequence identity of the CF is less than 100% compared to a specific sequence disclosed herein, the invention contemplates substitution of any of the other 19 natural L-amino acids for a given amino acid residue of the given CF (e.g., FIX or FVII), which may be at any position within the sequence of the CF, including adjacent amino acid residues. If any one substitution results in an undesirable change in biological activity, then one of the alternative amino acids can be employed and the construct evaluated by the methods described herein, or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934, the contents of which is incorporated by reference in its entirety, or using methods generally known in the art. In addition, variants can include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence of a CF that retains some if not all of the biological activity of the native peptide; e.g., the ability to activate another coagulation factor and/or participate in the coagulation cascade, leading to fibrin formation and hemostasis.
• In one embodiment, a factor IX incorporated into a CFXTEN fusion protein has a sequence that exhibits at least about 80% sequence identity compared to a sequence from Table 1, alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, sequence identity as compared with a sequence from Table 1.
• In one embodiment, a factor VII incorporated into a CFXTEN fusion protein has a sequence that exhibits at least about 80% sequence identity compared to a sequence from Table 2, alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, sequence identity as compared with a sequence from Table 2.

• TABLE 5
  Exemplary conservative amino acid substitutions

  	Original Residue	Exemplary Substitutions
  	Ala (A)	val; leu; ile
  	Arg (R)	lys; gln; asn
  	Asn (N)	gin; his; lys; arg
  	Asp (D)	Glu
  	Cys (C)	Ser
  	Gln (Q)	Asn
  	Glu (E)	Asp
  	Gly (G)	Pro
  	His (H)	asn: gin: lys: arg
  	xIle (I)	leu; val; met; ala; phe: norleucine
  	Leu (L)	norleucine: ile: val; met; ala: phe
  	Lys (K)	arg: gin: asn
  	Met (M)	leu; phe; ile
  	Phe (F)	leu: val: ile; ala
  	Pro (P)	Gly
  	Ser (S)	Thr
  	Thr (T)	Ser
  	Trp (W)	Tyr
  	Tyr (Y)	Trp: phe: thr: ser
  	Val (V)	Ile; leu; met; phe; ala; norleucine

1. Internal XTEN Sequences
• The present invention encompasses CFXTEN that comprise one or more XTEN sequences located internal to the CF sequence. The one or more internally-located XTEN can be a sequence length of 36 to ≧1000 amino acid residues. In some embodiments, the CFXTEN can have one or two or three or four or more XTEN sequences with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to one or more XTEN selected from Tables 4, 9, 10, 11, 12 and 13 wherein the XTEN sequences are located internal to the CF sequence. In one embodiment of the foregoing, the CFXTEN with one or more internal XTEN has an additional XTEN located at the N- or C-terminus of the fusion protein with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to one or more XTEN selected from Table 4. In another embodiment, the invention provided CFXTEN with internal XTEN (as detailed below) that further comprises a C-terminus XTEN linked to the CF by a cleavage sequence (e.g., a cleavage sequence of Table 7) such that the XTEN can be released when acted on by a protease. The linkage of XTEN by cleavage sequences is more fully described below and in the Examples.
• In some embodiments, as illustrated inFIG. 2 and described more fully in the Examples, an XTEN can be located between the domains of a FIX sequence; e.g., between the Gla and EGF1, or between the EGF1 and EGF2, or between the EGF2 and the activation peptide, or within the sequence of the activation peptide between the R145-A146 and R180-V181 activation peptide residues of the AP (i.e., between any two amino acids of the sequence TVFPDVDYVNSTEAETILDNITQSTQSFNDF (SEQ ID NO: 84)), or between the EGF2 and the activation peptide, or between the activation peptide and the protease domain, or any combination of the foregoing. In other embodiments, as illustrated inFIG. 2 and detailed more fully in the Examples, the XTEN can be inserted within an existing loop sequence within an individual domain of the FIX sequence so that 1) the XTEN forms a looped structure outside the domain and doesn't disrupt the normal architecture of the domain; and 2) the XTEN can be released by cleavage of incorporated cleavage sites.
• In another embodiment, the invention provides a CFXTEN comprising a FVII that incorporates one or more XTEN located between the domains of a FVII sequence; e.g., between the Gla and EGF1, or between the EGF1 and EGF2, or between the EGF2 and the activating peptide, or between the activating peptide and the protease domain, or any combination of the foregoing. The XTEN can be a sequence of 36 to >1000 amino acid residues including, but not limited to a sequence that has at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more sequence identity compared to a sequence from Table 4, 8, 9, 10, 11, 12, and 13. In one embodiment, as illustrated inFIGS. 5 and 6, an XTEN is incorporated between the EGF2 domain and the single lytic cleavage site at residues Arg¹⁵²-Ile¹⁵³. In other embodiments, as illustrated inFIGS. 5 and 6 and detailed more fully in the Examples, the XTEN can be inserted within an existing loop sequence within an individual domain of the FVII sequence so that 1) the XTEN forms a looped structure outside the domain and doesn't disrupt the normal architecture of the domain; and 2) the XTEN can be released by cleavage of incorporated cleavage sites.
2. Factor VII-FIX Hybrid Sequence Variants
• The invention provides an isolated factor VII polypeptide comprising at least one heterologous sequence that is cleavable by a pro-coagulant protease that does not activate a wildtype factor VII, wherein upon cleavage heterologous sequence, the factor VII polypeptide is activated. For example, CFXTEN with factor VII-factor IX hybrid sequence variants that incorporate into, or replace a portion of the sequence, a factor VII construct portions of the activating peptide domain (AP) sequence from factor IX, resulting in hybrid compositions that can be activated as part of the intrinsic system of the coagulation cascade. The CFXTEN that incorporate such factor VII-factor IX sequence variants as the CF component of the fusion protein permit administration to a subject a composition in which the CF component is not activated, and can be dosed at high amounts because it remains as an inert, circulating depot that is largely resistant to inactivation by protease inhibitors until activated by the triggering of the intrinsic coagulation cascade or by auto-activation, the latter a slow process. Non-limiting examples of FVII/FIX hybrid sequences are illustrated inFIG. 36, showing those portions of the hybrid amino acid sequences that have homology with those of native FIX and FVII. In some embodiments, the CFXTEN comprise factor VII-factor IX sequence variants that substitute portions or the entirety of the FIX activating peptide sequence with one or both FIX AP cleavage sites for FVII sequence to the N-terminal side of the protease domain of FVII; i.e., either towards the N-terminus beginning with the arginine at position 212 of the full-length precursor polypeptide or the isoleucine at position 213. In one embodiment, the factor VII-factor IX sequence CF incorporates the full-length FIX AP domain plus at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12 amino acids flanking adjacent amino acid residues on one or both sides of the R145-A146 and R180-V181 cleavage sites of FIX (e.g., the sequence RVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGE (SEQ ID NO: 1) in the case of 12 flanking amino acids on the N-terminus side and 5 flanking amino acids on the C-terminus side). In another embodiment, the CFXTEN comprises a factor VII-factor IX sequence variant that incorporates a portion of the AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 that flank the R145-A146 AP cleavage site (e.g., the sequence TSKLTRAETVFP (SEQ ID NO: 3) in the case of 6 flanking amino acids on either side of the cleavage site). In another embodiment, the CFXTEN comprises a factor VII-factor IX sequence variant that incorporates a portion of the AP that includes a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank one or both sides of the R180-V181 AP cleavage site (e.g., the sequence and DFTRV (SEQ ID NO: 4) in the case of 4 amino acids on the N-terminal flank and valine as the C-terminus of the cleavage site from FIX). In another embodiment, the CFXTEN comprises the factor VII-factor IX sequence variant of any of the foregoing embodiments of this paragraph that further includes the same AP sequence as a linker between the C-terminus of the FVII component and the XTEN component of the fusion protein; e.g., an N- to C-terminus configuration of FVII variant-AP sequence-XTEN, thereby permitting the release of the factor VII-factor IX sequence variant component from the CFXTEN fusion protein by the same intrinsic coagulation factor as per that of the FVII to FVIIa transition. In another embodiment, the CFXTEN comprises the factor VII-factor IX sequence variant of any of the foregoing embodiments of this paragraph that further includes the factor XI cleavage sequence KLTRAET (SEQ ID NO: 6) as the linker between the FVII variant sequence and the XTEN, thereby permitting the release of the factor VII-factor IX sequence variant component from the CFXTEN fusion protein by the initiation of the intrinsic coagulation cascade. It is expected d that with the release of the XTEN from the factor VII-factor IX sequence variant, the activated factor VII-factor IX sequence variant would have a shorter half-life compared to the intact CFXTEN, thereby increasing the margin of safety and tolerability of the composition in a subject. In the embodiments of the paragraph, the activated factor VII-factor IX sequence variant molecule can have at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the biological activity as native FVIIa, as measured by any of the appropriate assays or parameters disclosed herein (e.g., PT or bleeding time assays).
• In yet another embodiment, the invention provides the factor VII-factor IX sequence variants of the foregoing embodiments of this paragraph without a linked XTEN, permitting their administration to a subject as a circulating depot of the factor VII-factor IX hybrid that can be activated by either the intrinsic or extrinsic coagulation cascade. In one embodiment, the invention provides a CFXTEN with a factor VII-factor IX sequence variant with incorporated FIX-derived sequence with an overall sequence that exhibits at least about 80% sequence identity, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity compared to a sequence from Table 43. In another embodiment, the invention provides a factor VII-factor IX sequence variant with incorporated FIX-derived cleavage sequence (without an XTEN) with a sequence that exhibits at least about 80% sequence identity, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity as compared with a sequence from Table 43 without an XTEN.
• The CFXTEN comprising factor VII-factor IX sequence variants can be evaluated for biological activity using assays or in vivo parameters as described herein (e.g., in vitro coagulation assays or a pharmacodynamic effect in a hemophilia model), and those sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more activity compared to the corresponding native FVII sequence is considered suitable for inclusion in the subject CFXTEN. The CF found to retain a suitable level of activity can be linked to one or more XTEN polypeptides described hereinabove. In one embodiment, a CF found to retain a suitable level of activity can be linked to one or more XTEN polypeptides having at least about 80% sequence identity to a sequence from Table 4, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared with a sequence of Table 4, resulting in a chimeric fusion protein.
3. CFXTEN Fusion Protein Configurations
• The invention provides CFXTEN fusion protein compositions with the CF and XTEN components linked in specific N- to C-terminus configurations. In some embodiments, one or more CFs are linked to one or more XTENs, either at the N-terminus or at the C-terminus, with or without a spacer, to form a block copolymer, and the sequential arrangement of the CFs and the XTENs in the CFXTEN fusion protein are the same as the configuration known in the block copolymer chemistry. When there is more than one CF, XTEN, or spacer, each of the CF, the XTEN, or the spacer have the same or different sequences, and the CFs and/or XTENs are linked either continuously or alternately (regular or irregular). Thus, in all of the formulae provided herein, when there is more than one CF, XTEN, or spacer, each of the CF, XTEN, and spacer are the same or different. In some embodiments, the CFXTEN is a monomeric fusion protein with a CF linked to one XTEN polypeptide. In other embodiments, the CFXTEN is a monomeric fusion protein with a CF linked to two or more XTEN polypeptides. In still other embodiments, the CFXTEN is a monomeric fusion protein with two or more CF linked to one XTEN polypeptide. In still other embodiments, the CFXTEN is a monomeric fusion protein with two or more CF linked to two or more XTEN polypeptide. In still other embodiment, the CFXTEN is a monomeric fusion protein with a single CF in which XTEN is located within the CF sequence (e.g., within a FIX sequence such as between one or more domains as illustrated inFIGS. 2 and 5). Table 6 provides non-limiting examples of configurations that are encompassed by the CFXTEN fusion proteins of the invention; numerous other variations will be apparent to the ordinarily skilled artisan, including the incorporation the spacer and cleavage sequences disclosed herein or known in the art.

• TABLE 6
  CFXTEN configurations

  	Components*	Configuration**
  	Single CF; Single XTEN	CF-XTEN
  		XTEN-CF
  	Single CF; Multiple XTEN	XTEN-CF-XTEN
  		CF-XTEN-XTEN
  		XTEN-XTEN-CF
  		XTEN-CF-XTEN-XTEN
  		XTEN-XTEN-CF-XTEN
  		XTEN-XTEN-CF-XTEN
  	Multiple CF, Single XTEN	CF-XTEN-CF
  		XTEN-CF-CF
  		CF-CF-XTEN
  		CF-XTEN-CF-CF
  	Multiple CF; Multiple XTEN	CF-XTEN-CF-XTEN
  		XTEN-CF-XTEN-CF
  		XTEN-XTEN-CF-XTEN-CF
  		XTEN-XTEN-CF-CF
  		CF-XTEN-XTEN-CF
  		CF-CF-XTEN-XTEN
  		CF-CF-XTEN-XTEN-CF
  		CF-XTEN-CF-XTEN-CF
  	*Characterized as single for 1 component or multiple for 2 or more of that component
  	**Reflects N- to C-terminus configuration of the growth factor and XTEN components

• The invention contemplates CFXTEN fusion proteins compositions comprising, but not limited to single or multiple CF selected from Table 1 or Table 2 (or fragments or sequence variants thereof), single or multiple XTEN selected from Table 4 (or sequence variants thereof) that are in a configuration shown in Table 6. Non-limiting examples of sequences of fusion proteins containing a single CF linked to a single XTEN are presented in Table 41. In one embodiment, a CFXTEN composition would comprise a fusion protein having at least about 80% sequence identity compared to a CFXTEN from Table 41, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 41. Generally, the resulting CFXTEN retains at least a portion of the biological activity of the corresponding CF not linked to the XTEN. In the foregoing fusion proteins hereinabove described in this paragraph, the CFXTEN fusion protein can further comprise a cleavage sequence from Table 7; the cleavage sequence being located between the CF and the XTEN or between adjacent CF (if more than one CF is included in the CFXTEN). In some cases, the CFXTEN comprising the cleavage sequences will also have one or more spacer sequence amino acids between the CF and the cleavage sequence or the XTEN and the cleavage sequence to facilitate access of the protease; the spacer amino acids comprising any natural amino acid, including glycine and alanine as preferred amino acids. Non-limiting examples of CFXTEN comprising CF, XTEN, cleavage sequence(s) and spacer amino acids are presented in Table 42. However, the invention also contemplates substitution of any of the CF sequences of Tables 1 and 2 for a CF sequence of Table 42, substitution of any XTEN sequence of Table 4 for an XTEN sequence of Table 42, and substitution of any cleavage sequence of Table 7 for a cleavage sequence of Table 42. In CFXTEN embodiments having one or more cleavage sequences, the CF component either becomes biologically active or has an increase in activity upon its release from the XTEN by cleavage of the cleavage sequence(s), described more fully below.
• In one embodiment of the CFXTEN composition, the invention provides a fusion protein of formula I:
• 
  (XTEN)_x-CF-(XTEN)_y  I
• wherein independently for each occurrence, CF is a coagulation factor; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides a fusion protein of formula II:
• 
  (XTEN)_x-(CF)-(S)_y-(XTEN)_y  II
• wherein independently for each occurrence, CF is a coagulation factor a; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein, wherein the fusion protein is of formula III:
• 
  (XTEN)_x-(S)_x-(CF)-(S)_y-(XTEN)_y  III
• wherein independently for each occurrence, CF is a coagulation factor; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula IV:
• 
  (Gla)-(XTEN)_u-(EGF1)-(XTEN)_v-(EGF2)-(XTEN)_w-(AP)-(XTEN)_x-(Pro)-(S)_y-(XTEN)_z  IV
• wherein independently for each occurrence, Gla is a Gla domain of FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP is an activator peptide of FIX; PRO is a protease domain of FIX; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1, z is either 0 or 1, with the proviso that u+v+x+z≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula V:
• 
  (Gla)-(XTEN)_u(EGF1)-(XTEN)_v-(EGF2)-(AP1)-(XTEN)_w-(AP2)-(XTEN)_x-(Pro)-(S)_y-(XTEN)_z  V
• wherein independently for each occurrence, Gla is a Gla domain of FIX; EGF1 is an EGF1 domain of FIX; EGF2 is an EFG2 domain of FIX; AP1 is the N-terminal sequence portion of the activator peptide domain of FIX that includes a first native cleavage sequence of the AP domain; AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes a second native cleavage sequence of the AP domain; PRO is a protease domain of FIX; S is a spacer sequence having between Ito about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+z≧1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula VI:
• 
  (Gla)-(XTEN)_u-(EGF1)-(XTEN)_v-(EGF2)-(XTEN)_w-(Pro)-(S)_x—(XTEN)_y  VI
• wherein independently for each occurrence, Gla is a Gla domain of FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is a protease domain of FVII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; and XTEN is an extended recombinant polypeptide.
• In another embodiment of the CFXTEN composition, the invention provides an isolated fusion protein of formula VII:
• 
  (Gla)-(XTEN)_t-(EGF1)-(XTEN)_u-(EGF2)-(AP1)_v-(XTEN)_w-(AP2)_x-(Pro)-(S)_y-(XTEN)_z  VII
• wherein independently for each occurrence, Gla is a Gla domain of FVII; EGF1 is an EGF1 domain of FVII; EGF2 is an EFG2 domain of FVII; PRO is a protease domain of FVII; AP1 is the N-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence; AP2 is the C-terminal sequence portion of the activator peptide domain of FIX that includes the native cleavage sequence; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; x is either 0 or 1; y is either 0 or 1; z is either 0 or 1; and XTEN is an extended recombinant polypeptide. In the embodiment, the factor VII variant includes can include one or both cleavage sequences from the activator peptide domain of factor IX; e.g., a sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank the R145-A146 cleavage site (e.g., the sequence TSKLTRAETVFP (SEQ ID NO: 3) in the case of 5 flanking amino acids) and the sequence of at least about 2, or at least about 3, or at least about 4, or at least about 5 amino acids that flank the R180-V181 cleavage site (e.g., the sequence FNDFTRVVGGED (SEQ ID NO: 85) in the case of 5 flanking amino acids, as described more fully above. The invention also contemplates substitution of any of the other cleavage sequences of Table 7 for the AP sequences of the factor VII variant.
• The embodiments of formulae V and VI encompass CFXTEN configurations of factor IX and factor VII, respectively, wherein one or more XTEN of lengths ranging from about 36 amino acids to ≧1000 amino acids (e.g., sequences selected from Tables 4, and 9-13) are inserted and linked between adjoining domains of the factor IX or the factor VII sequence, respectively. The invention contemplates all possible permutations of insertions of XTEN between the domains of either FIX or FVII with optional linking of an additional XTEN to the C-terminus of the FIX or the FVII, optionally via an additional cleavage sequence selected from Table 7, resulting in a CFXTEN composition; non-limiting examples of which are portrayed inFIGS. 2,5 and6. In the foregoing embodiments hereinabove described in this paragraph, the CFXTEN fusion proteins can be evaluated for retention of biological activity (including after cleavage of any incorporated XTEN-releasing cleavage sites) using any appropriate in vitro assay disclosed herein (e.g., the assays of Table 40 or the assays described in the Examples), to determine the suitability of the configuration for use as a therapeutic agent in the treatment of a coagulation-factor related disease, disorder or condition.
• In some embodiments, administration of a therapeutically effective amount of a fusion protein of one of formulae I-VII to a subject in need thereof results in an increase of at least two-fold in the terminal half-life, or at least three-fold, or at least four-fold, or at least five-fold, or at least 10-fold, or at least 20-fold, or at least 40-fold, or at least 100-fold increase in the terminal half-life for the fusion protein compared to the corresponding CF not linked to the XTEN and administered at a comparable amount administered to a subject. In some embodiments, administration of a therapeutically effective amount of a fusion protein of one of formulae I-VII to a subject in need thereof results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least 10-fold, or at least 20-fold, or at least 40-fold, or at least 100-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding CF not linked to the XTEN and administered at a comparable amount administered to a subject. In other embodiments, administration of a therapeutically effective dose of a fusion protein of one of formulae I-VII to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain a therapeutically effective blood level of the fusion protein of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to a CF not linked to XTEN and administered at a comparable dose.
• Any spacer sequence group optionally is introduced to a subject fusion protein encompassed by the invention. The spacer is provided to enhance expression of the fusion protein from a host cell or to decrease steric hindrance such that the CF component may assume its desired tertiary structure and/or interact appropriately with its target substrate. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated by reference herein. In one embodiment, the spacer comprises one or more peptide sequences that are between 1-50 amino acid residues in length, or about 1-25 residues, or about 1-10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). In some cases, the spacer can be polyglycines or polyalanines, or is predominately a mixture of combinations of glycine and alanine residues. The spacer polypeptide exclusive of a cleavage sequence is largely to substantially devoid of secondary structure; e.g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/or GOR algorithms. In one embodiment, a spacer sequence in a CFXTEN fusion protein composition further contains one or more cleavage sequences, which are identical or different, wherein the cleavage sequence may be acted on by a protease to release the CF from the fusion protein.
• In some embodiments, the incorporation of the cleavage sequence into the CFXTEN is designed to permit release of a CF that becomes active or more active upon its release from the XTEN; e.g., the enzymatic activity of the CF component increases. In one embodiment of the foregoing, the CF that becomes active after release is a FIX or a sequence variant thereof. In another embodiment of the foregoing, the CF that becomes active after release is a FVII or a sequence variant thereof. The cleavage sequences are located sufficiently close to the CF sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the CF sequence terminus, such that any remaining residues attached to the CF after cleavage do not appreciably interfere with the activity (e.g., such as binding to a ligand or substrate) of the CF, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the CFXTEN can be cleaved after administration to a subject. In such cases, the CFXTEN can serve as a prodrug or a circulating depot for the CF. In one embodiment, the CF that is released from the fusion protein by cleavage of the cleavage sequence exhibits at least about a two-fold, or at least about a three-fold, or at least about a four-fold, or at least about a five-fold, or at least about a six-fold, or at least about a eight-fold, or at least about a ten-fold, or at least about a 20-fold increase in enzymatic activity for its native substrate compared to the intact CFXTEN fusion protein.
• Examples of cleavage sites contemplated by the invention include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases such as TEV, enterokinase, PreScission™ protease (rhinovirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases and others are known in the art. Exemplary cleavage sequences and cut sites within the sequences are presented in Table 7, as well as sequence variants thereof. For example, thrombin (activated clotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 86) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], which is cut after the arginine atposition 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor IX in the coagulation pathway. Once activated, its natural role in coagulation is to cleave fibrinogen, which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. However, as coagulation is an on-going process in mammals, by incorporation of the LTPRSLLV (SEQ ID NO: 86) sequence into the CFXTEN between and linking the CF and the XTEN components, the XTEN is removed from the adjoining CF concurrent with activation of either the extrinsic or intrinsic coagulation pathways when coagulation is required physiologically, thereby releasing CF over time. Similarly, incorporation of other cleavage sequences into CFXTEN that are acted upon by endogenous proteases, particularly those susceptible to the activated clotting proteins listed in Table 7, would provide for sustained release of CF that, in certain embodiments of the CFXTEN, provide a higher degree of activity for the CF component released from the intact form of the CFXTEN. In one embodiment, the invention provides CFXTEN comprising one or more cleavage sequences operably positioned to release the CF from the fusion protein upon cleavage, wherein the one or more cleavage sequences has at least about 86%, or at least about 92% or greater sequence identity to a sequence selected from Table 7. In another embodiment, the CFXTEN comprising a cleavage sequence would have at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity compared to a sequence selected from Table 42.
• In some embodiments, only the two or three amino acids flanking both sides of the cut site (four to six amino acids total) are incorporated into the cleavage sequence that, in turn, is incorporated into the CFXTEN of the embodiments. In other embodiments, the known cleavage sequence have one or more deletions or insertions or one or two or three amino acid substitutions for any one or two or three amino acids in the known sequence, wherein the deletions, insertions or substitutions result in reduced or enhanced susceptibility but not an absence of susceptibility to the protease, resulting in an ability to tailor the rate of release of the CF from the XTEN. Exemplary substitutions are shown in Table 7.

• TABLE 7
  Protease Cleavage Sequences

  	Exemplary
  Protease Acting	Cleavage	SEQ ID		SEQ ID
  Upon Sequence	Sequence	NO:	Minimal Cu Site*	NO:

  FXIa	KLTR↓AET	87	KD/FL/T/R↓VA/VE/GT/GV
  FXIa	DFTR↓VVG	88	KD/FL/T/R↓VA/VE/GT/GV
  FXIIa	TMTR↓IVGG	89	NA
  Kallikrein	SPFR↓STGG	90	—/—/FL/RY↓SR/RT/—/—
  FVIIa	LQVR↓IVGG	91	NA
  FIXa	PLGR↓IVGG	92	—/—/G/R↓—/—/—/—
  FXa	IEGR↓TVGG	93	IA/E/GFP/R↓STI/VFS/—/G
  FIIa (thrombin)	LTPR↓SLLV	94	—/—/PLA/R↓SAG/—/—/—
  Elastase-2	LGPV↓SGVP	95	—/—/—VIAT↓—/—/—/—
  Granzyme-B	VAGD↓SLEE	96	V/—/—/D↓—/—/—/—
  MMP-12	GPAG↓LGGA	97	G/PA/—/G↓L/—/G/—	98
  MMP-13	GPAG↓LRGA	99	G/P/—/G↓L/—/GA/—	100
  MMP-17	APLG↓LRLR	101	—/PS/—/—↓LQ/—/LT/—
  MMP-20	PALP↓LVAQ	102	NA
  TEV	ENLYFQ↓G	103	ENLYFQ↓G/S	104
  Enterokinase	DDDK↓IVGG	105	DDDK↓IVGG	106
  Protease 3C	LEVLFQ↓GP	107	LEVLFQ↓GP	108
  (PreScission ™)
  Sortase A	LPKT↓GSES	109	L/P/KEAD/T↓G/—/EKS/S	110
  ↓ indicates cleavage site
  NA: not applicable
  *the listing of multiple amino acids before, between, or after a slash indicate alternative amino acids that can be substituted at the position;
  “—” indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column

(a) Pharmacokinetic Properties of CFXTEN
• The invention provides CFXTEN fusion proteins with enhanced pharmacokinetics compared to the CF not linked to XTEN. The pharmacokinetic properties of a CF that can be enhanced by linking a given XTEN to the CF include, but are not limited to, terminal half-life, area under the curve (AUC), C_max, volume of distribution, and bioavailability; properties that provide enhanced utility in the treatment of coagulation factor-related disorders, diseases and related conditions. As a result of the enhanced properties, the CFXTEN, when used at the dose and dose regimen determined to be appropriate for the composition by the methods described herein, can achieve a circulating concentration resulting in a desired pharmacologic effect, yet stay within the safety range for biologically active component of the composition for an extended period of time compared to a comparable dose of the CF not linked to XTEN. In such cases, the CFXTEN remains within the therapeutic window for the fusion protein composition for the extended period of time compared to a CF not liked to XTEN and administered to a subject at a comparable dose. As used herein, a “comparable dose” means a dose with an equivalent moles/kg for the active CF pharmacophore (e.g., FIX or FVII) that is administered to a subject in a comparable fashion. It will be understood in the art that a “comparable dosage” of CFXTEN fusion protein would represent a greater weight of agent but would have essentially the same mole-equivalents of CF in the dose of the fusion protein administered.
• In some embodiments, the CFXTEN with enhanced pharmacokinetic properties can be a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a protein sequence selected from any one of Tables 41, 42, or 43. In other embodiments, the CFXTEN with enhanced pharmacokinetic properties can comprise a CF sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 1 or from Table 2, linked to one or more XTEN that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity compared to a sequence from Table 4. For the inventive compositions, CFXTEN with a longer terminal half-life are generally preferred, so as to improve patient convenience, to increase the interval between doses and to reduce the amount of drug required to achieve a sustained effect. In the embodiments hereinabove described in this paragraph the administration of the fusion protein results in an improvement in at least one of the parameters (disclosed herein as being useful for assessing the subject diseases, conditions or disorders) using a lower unit dose in moles of fusion protein compared to the corresponding CF component not linked to the fusion protein and administered at a comparable unit dose or dose regimen to a subject. In the foregoing embodiments, the total dose in moles administered to achieve the improvement is at least about three-fold lower, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding CF component not linked to the fusion protein.
• As described more fully in the Examples pertaining to pharmacokinetic characteristics of fusion proteins comprising XTEN, it was observed that increasing the length of the XTEN sequence confers a disproportionate increase in the terminal half-life of a fusion protein comprising the XTEN. Accordingly, the invention provides CFXTEN fusion proteins comprising XTEN wherein the XTEN is selected to provide a targeted half-life for the CFXTEN composition administered to a subject. In some embodiments, the invention provides monomeric fusion proteins comprising XTEN wherein the XTEN is selected to confer an increase in the terminal half-life for the CFXTEN administered to a subject, compared to the corresponding CF not linked to the fusion protein and administered at a comparable dose, wherein the increase is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least a 40-fold, or at least a 80-fold, or at least a 100-fold or greater an increase in terminal half-life compared to the CF not linked to the fusion protein. Exogenously administered factor IX has been reported to have a terminal half-life in humans of approximately 18-24 hours (Morfini, M. Blood Transfus. (2008) 6(s2): s21-s25) and exogenously administered factor VII is reported to have a terminal half-life of approximately 4-6 hours (Klitgaard T, Br J Clin Pharmacol (2008) 65(1):3-11), where as various CFXTEN compositions disclosed herein that have been experimentally administered to animals, as described in the Examples, have resulted in terminal half-life values considerably longer. In one embodiment, the present invention provides CFXTEN fusion proteins that exhibits an increase in ACU of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000% compared to the corresponding CF not linked to the XTEN and administered to a subject at a comparable dose. The pharmacokinetic parameters of a CFXTEN can be determined by standard methods involving dosing, the taking of blood samples at times intervals, and the assaying of the protein using ELISA, HPLC, radioassay, or other methods known in the art or as described herein, followed by standard calculations of the data to derive the half-life and other PK parameters.
• The enhanced PK parameters allow for reduced dosing of the CFXTEN compositions, compared to CF not linked to XTEN. In some embodiments, a smaller molar amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold less or greater of the fusion protein is administered in comparison to the corresponding CF not linked to the XTEN under a dose regimen needed to maintain hemostasis, and the fusion protein achieves a comparable area under the curve as the corresponding molar amount of the CF not linked to the XTEN. In other embodiments, the fusion protein has a less frequent administration regimen of about every two days, about every seven days, about every 14 days, about every 21 days, or about monthly of the fusion protein administered to a subject, compared to the daily administration of an otherwise same dose amount of the corresponding CF not linked to the XTEN, and the fusion protein achieves a comparable area under the curve as the corresponding CF not linked to the XTEN. In yet other embodiments, an accumulative smaller molar amount of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered to a subject in comparison to the corresponding molar amount of the CF not linked to the XTEN under a dose regimen needed to maintain hemostasis, yet the fusion protein achieves at least a comparable area under the curve as the corresponding CF not linked to the XTEN. The accumulative smaller molar amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month.
• The invention further provides CFXTEN comprising a CF molecule separated from the XTEN sequence by one or more cleavage sequences; e.g., a sequence from Table 7. In some embodiments, the intact CFXTEN composition has less activity but a longer half-life in its intact form compared to a corresponding CF not linked to the XTEN, but is designed such that upon administration to a subject, the CF component is gradually released from the fusion protein by cleavage at the cleavage sequence(s) by endogenous proteases, whereupon the CF component exhibits activity, i.e., the ability to effectively bind to and activate its target coagulation protein substrate. In non-limiting examples, the CFXTEN with a cleavage sequence has about 80% sequence identity compared to a sequence from Table 42, or about 85%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence identity compared to a sequence from Table 42. Accordingly, the CFXTEN of the foregoing embodiments in this paragraph serve as prodrugs or a circulating depot, resulting in a longer terminal half-life compared to CF not linked to the XTEN. In such cases, a higher concentration of CFXTEN can be administered to a subject, compared to the corresponding CF not linked to XTEN because a smaller proportion of the circulating composition is active.
(b) Pharmacology and Pharmaceutical Properties of CFXTEN
• The present invention provides CFXTEN compositions comprising CF covalently linked to XTEN that can have enhanced properties compared to CF not linked to XTEN, as well as methods to enhance the therapeutic and/or biologic activity or effect of the respective two CF components of the compositions. In addition, the invention provides CFXTEN compositions with enhanced properties compared to those art-known fusion proteins containing albumin, immunoglobulin polypeptide partners, polypeptides of shorter length and/or polypeptide partners with repetitive sequences. In addition, CFXTEN fusion proteins provide significant advantages over chemical conjugates, such as pegylated constructs, notably the fact that recombinant CFXTEN fusion proteins can be made in bacterial cell expression systems, which can reduce time and cost at both the research and development and manufacturing stages of a product, as well as result in a more homogeneous, defined product with less toxicity for both the product and metabolites of the CFXTEN compared to pegylated conjugates.
• As therapeutic agents, the CFXTEN possesses a number of advantages over therapeutics not comprising XTEN, including one or more of the following non-limiting exemplary enhanced properties: increased solubility, increased thermal stability, reduced immunogenicity, increased apparent molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efficiency, a lower effective therapeutic dose, increased bioavailability, increased time between dosages capable of maintaining blood levels within the therapeutic window for the CF, a “tailored” rate of absorption when administered subcutaneously or intramuscularly, enhanced lyophilization stability, enhanced serum/plasma stability, increased terminal half-life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased active clearance, reduced side effects, retention of substrate binding affinity, stability to degradation, stability to freeze-thaw, stability to proteases, stability to ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased stability in storage (e.g., increased shelf-life), reduced toxicity in an organism or environment and the like. The net effect of the enhanced properties is that the use of a CFXTEN composition can result in enhanced therapeutic and/or biologic effect compared to a CF not linked to XTEN or result in improved patient compliance when administered to a subject with a coagulation factor-related disease or disorder.
• Specific assays and methods for measuring the physical and structural properties of expressed proteins are known in the art, including methods for determining properties such as protein aggregation, solubility, secondary and tertiary structure, melting properties, contamination and water content, etc. Such methods include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy; several of which are applied to the inventive CFXTEN as described in the Examples. Additional methods are disclosed in Arnau et al, Prot Expr and Purif (2006) 48, 1-13. Application of these methods to elucidate the enhanced properties of the compositions of the invention is within the grasp of a person skilled in the art.
• In one embodiment, XTEN as a fusion partner increases the solubility of the CF payload. Accordingly, where enhancement of the pharmaceutical or physicochemical properties of the CF is desirable, such as the degree of aqueous solubility or stability, the length and/or the motif family composition of the XTEN sequences incorporated into the fusion protein may each be selected to confer a different degree of solubility and/or stability on the respective fusion proteins such that the overall pharmaceutical properties of the CFXTEN composition are enhanced. The CFXTEN fusion proteins can be constructed and assayed, using methods described herein, to confirm the physicochemical properties and the XTEN adjusted, as needed, to result in the desired properties. In one embodiment, the XTEN sequence of the CFXTEN is selected such that the fusion protein has an aqueous solubility that is within at least about 25% greater compared to a CF not linked to the fusion protein, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% greater than the corresponding CF not linked to the fusion protein.
• The invention provides methods to produce and recover expressed CFXTEN from a host cell with enhanced solubility and ease of recovery compared to CF not linked to XTEN. In some embodiments, the method includes the steps of transforming a eukaryotic host cell with a polynucleotide encoding a CFXTEN with one or more XTEN components of cumulative sequence length greater than about 200, or greater than about 400, or greater than about 600, or greater than about 800 amino acid residues, expressing the CFXTEN fusion protein in the host cell, and recovering the expressed fusion protein in soluble form. In the embodiments hereinabove described in this paragraph, the XTEN of the CFXTEN fusion proteins can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN selected from Table 4 and the CF can have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity compared to a CF selected from Table 1 or Table 2 and the CFXTEN components can be in an N- to C-terminus configuration selected from formulas I-VII.
• In one embodiment, the invention provides CFXTEN compositions and methods to produce the compositions that can maintain the CF component within a therapeutic window for a greater period of time compared to comparable dosages of the corresponding CF not linked to XTEN. It will be understood in the art that a “comparable dosage” of CFXTEN fusion protein would represent a greater weight of agent but would have the same approximate mole-equivalents of CF in the dose of the fusion protein and/or would have the same approximate molar concentration relative to the CF. The method to produce the compositions that can maintain the CF component within a therapeutic window includes the steps of selecting the XTEN appropriate for conjugation to a CF to provide the desired pharmacokinetic properties in view of a given dose and dose regimen, administration of the CFXTEN to subjects in need thereof, followed by assays to verify the pharmacokinetic properties, the activity of the CFXTEN fusion protein, and the safety of the administered composition. By the methods, CFXTEN provided herein allow for increased efficacy of the administered composition by maintaining the circulating concentrations of the CF within the therapeutic window for an enhanced period of time. As used herein, “therapeutic window” means that the amount of drug or biologic as a blood or plasma concentration range, which provides efficacy or a desired pharmacologic effect over time for the disease or condition without unacceptable toxicity, i.e., the range of the circulating blood concentrations between the minimal amount to achieve any positive therapeutic effect and the maximum amount which results in a response that is the response immediately before toxicity to the subject (at a higher dose or concentration). Additionally, therapeutic window generally encompasses an aspect of time; the maximum and minimum concentration that results in a desired pharmacologic effect over time that does not result in unacceptable toxicity or adverse events. A dosed composition that stays within the therapeutic window for the subject could also be said to be within the “safety range.”
• The characteristics of CFXTEN compositions of the invention, including functional characteristics or biologic and pharmacologic activity and parameters that result, can be determined by any suitable screening assay known in the art for measuring the desired characteristic. The invention provides methods to assay the CFXTEN fusion proteins of differing composition or configuration in order to provide CFXTEN with the desired degree of biologic and/or therapeutic activity, as well as safety profile. Specific in vivo and ex vivo biological assays are used to assess the activity of each configured CFXTEN and/or CF component to be incorporated into CFXTEN, including but not limited to the assays of the Examples, those assays of Table 40, as well as the following assays or other such assays known in the art for assaying the properties and effects of CF. Functional assays can be conducted that allow determination of coagulation activity, such as prothrombin (PT) and activated partial prothrombin (aPTT) assays (Belaaouaj A A et al., J. Biol. Chem. (2000) 275:27123-8; Diaz-Collier J A. Haemost (1994) 71:339-46), blood clotting time (WBCT), thrombelastography, or bleeding time assays. Other possible assays may determine the binding affinity of a CFXTEN for the target substrate of the corresponding CF can be assayed using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in U.S. Pat. No. 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. The foregoing assays can also be used to assess CF sequence variants (assayed as single components or as CFXTEN fusion proteins) and can be compared to the native CF to determine whether they have the same degree of biologic activity as the native CF, or some fraction thereof such that they are suitable for inclusion in CFXTEN; e.g., at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native CF.
• Dose optimization is important for all drugs, especially for those with a narrow therapeutic window. For example, a standardized single dose of CF for all patients presenting with a diverse symptoms or abnormal clinical parameters may not always be effective. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically or pharmacologically effective amount of the CFXTEN, versus that amount that would result in unacceptable toxicity and place it outside of the safety range, or insufficient potency such that clinical improvement is not achieved.
• In many cases, the therapeutic window for CF in subjects of different ages or degree of disease have been established and are available in published literature or are stated on the drug label for approved products containing the CF. In other cases, the therapeutic window can be established for new compositions, including those CFXTEN of the disclosure. The methods for establishing the therapeutic window for a given composition are known to those of skill in the art (see, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11^thEdition, McGraw-Hill (2005)). For example, by using dose-escalation studies in subjects with the target disease or disorder to determine efficacy or a desirable pharmacologic effect, appearance of adverse events, and determination of circulating blood levels, the therapeutic window for a given subject or population of subjects can be determined for a given drug or biologic, or combinations of biologics or drugs. The dose escalation studies can evaluate the activity of a CFXTEN through metabolic studies in a subject or group of subjects that monitor physiological or biochemical parameters, as known in the art or as described herein for one or more parameters associated with the metabolic disease or disorder, or clinical parameters associated with a beneficial outcome for the particular indication, together with observations and/or measured parameters to determine the no effect dose, adverse events, maximum tolerated dose and the like, together with measurement of pharmacokinetic parameters that establish the determined or derived circulating blood levels. The results can then be correlated with the dose administered and the blood concentrations of the therapeutic that are coincident with the foregoing determined parameters or effect levels. By these methods, a range of doses and blood concentrations can be correlated to the minimum effective dose as well as the maximum dose and blood concentration at which a desired effect occurs and above which toxicity occurs, thereby establishing the therapeutic window for the dosed therapeutic. Blood concentrations of the fusion protein (or as measured by the CF component) above the maximum is considered outside the therapeutic window or safety range. Thus, by the foregoing methods, a C_minblood level is established, below which the CFXTEN fusion protein would not have the desired pharmacologic effect, and a C_maxblood level is established that would represent the highest circulating concentration before reaching a concentration that would elicit unacceptable side effects, toxicity or adverse events, placing it outside the safety range for the CFXTEN. With such concentrations established, the frequency of dosing and the dosage can be further refined by measurement of the C_maxand C_minto provide the appropriate dose and dose frequency to keep the fusion protein(s) within the therapeutic window.
• One of skill in the art can, by the means disclosed herein or by other methods known in the art, confirm that the administered CFXTEN remains in the therapeutic window for the desired interval or requires adjustment in dose or length or sequence of XTEN. Further, the determination of the appropriate dose and dose frequency to keep the CFXTEN within the therapeutic window establishes the therapeutically effective dose regimen; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof resulting in consecutive C_maxpeaks and/or C_mintroughs that remain within the therapeutic window and results in an improvement in at least one measured parameter relevant for the target disease, disorder or condition. In some cases, the CFXTEN administered at an appropriate dose to a subject results in blood concentrations of the CFXTEN fusion protein that remains within the therapeutic window for a period at least about two-fold longer compared to the corresponding CF not linked to XTEN and administered at a comparable dose; alternatively at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer or greater compared to the corresponding CF not linked to XTEN and administered at a comparable dose. As used herein, an “appropriate dose” means a dose of a drug or biologic that, when administered to a subject, would result in a desirable therapeutic or pharmacologic effect and/or a blood concentration within the therapeutic window.
• In one embodiment, the CFXTEN administered at a therapeutically effective dose regimen results in a gain in time of at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive C_maxpeaks and/or C_mintroughs for blood levels of the fusion protein compared to the corresponding biologically active protein of the fusion protein not linked to the fusion protein and administered at a comparable dose regimen to a subject. In another embodiment, the CFXTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameter using less frequent dosing or a lower total dosage in moles of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the fusion protein and administered to a subject using a therapeutically effective dose regimen for the CF. The measured parameters include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with coagulation factor-related disorders.
• In some embodiments, the CFXTEN fusion proteins of the invention retain at least about 0.05%, or about 0.1%, or about 1%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 98%, or about 99% percent of the biological activity of the corresponding CF not linked to the fusion protein with regard to an in vitro biologic activity or pharmacologic effect known or associated with the use of the native CF in the treatment and prevention of coagulation factor-related diseases, disorders, and conditions. Non-limiting examples of parameters or physiologic effects that can be assayed to assess the retained activity of the CFXTEN fusion proteins include prothrombin time (PT), activated partial thromboplastin time (aPTT), bleeding time, whole blood clotting time (WBCT), and thrombelastography. In some embodiments, the activity of the CF component is manifested by the intact CFXTEN fusion protein, while in other cases the activity of the CF component is primarily manifested upon cleavage and release of the CF from the fusion protein by action of a protease that acts on a cleavage sequence incorporated into the CFXTEN fusion protein, embodiments of which are disclosed above. In the foregoing, the CFXTEN is designed to reduce the binding affinity of the CF component for the coagulation substrate when linked to the XTEN but have restored or increased affinity when released from XTEN through the cleavage of cleavage sequence(s) incorporated into the CFXTEN sequence, as described more fully above. In one embodiment of the foregoing, the invention provides an isolated fusion protein comprising a FIX linked to XTEN by a cleavage sequence, wherein the fusion protein is substantially inactive prior to cleavage and wherein the FIX released from the fusion protein by proteolytic cleavage at the cleavage sequence has biological activity that is at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% as active compared to native FIX not linked to XTEN.
• In other cases, the CFXTEN can be designed to reduce active clearance of the CFXTEN to increase the terminal half-life of CFXTEN administered to a subject, while still retaining biological activity. The clearance mechanisms to remove CF from the circulation have yet to be fully elucidated. Uptake, elimination, and inactivation of CFs can occur in the circulatory system as well as in the extravascular space. Coagulation factors are complex proteins that interact with a large number of other proteins, lipids, and receptors, and many of these interactions can contribute to the elimination of CFs from the circulation. For example, clearance mechanisms for FVII, a heterogeneously glycosylated protein, may include clearance by the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on FVIIa clearance have been investigated using a perfused liver model, with results suggesting that carbohydrate receptors (e.g. the asialoglycoprotein receptor, ASGPR) may play a role in FVIIa clearance. (Appa, R. S., et al. Thromb Haemost. (2010, epub May 27) 104(2)). In addition, CF can be lost through extravasation and rapid active clearance, which is reflected in the generally poor bioavailability of intravenously administered coagulation factors such as factor VIIa (see NovoSeven package insert). It is believed that the CFXTEN of the present invention has comparatively higher bioavailability achieved by reduced active clearance and/or by reduced extravasation by increasing the hydrodynamic radius, or apparent size, of the molecule by the addition of unstructured XTEN to the coagulation factor. In one embodiment, the invention provides CFXTEN that reduce clearance of the fusion protein by linking one or more XTEN to the CF component of the fusion protein, wherein the fusion protein has an increase in apparent molecular weight factor of at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about ten-fold, or at least about twelve-fold, or at least about fifteen-fold, and wherein the terminal half-life of the CFXTEN when administered to a subject is increased at least about two-fold, or at least about four-fold, or at least about eight-fold, or at least about 10-fold, or at least about 20-fold, or at least about 30-fold, or at least about 40-fold, or at least about 50-fold, or at least about 60-fold, or at least about 70-fold, or at least about 80-fold or more compared to the corresponding CF not linked to XTEN. In the foregoing embodiment, wherein at least two XTEN molecules are incorporated into the CFXTEN, the XTEN can be identical or they can be of a different sequence composition (and net charge) or length. Non-limiting examples of the foregoing embodiment with two XTEN linked to a single FVII are illustrated inFIG. 6, and include the constructs (expressed using the domains of FVII) Gla-EGF1-EGF2-AE144-Protease-AE864 or Gla-EGF1-AE288-EGF2-Protease-AE864 (wherein the AE XTEN components have approximately a 17% net charge due to incorporated glutamic acid), Gla-EGF1-EGF2-AG144-Protease-AG864 or Gla-EGF1-AG144-EGF2-Protease-AE864 (wherein the AG XTEN components have approximately no net charge). Not to be bound by a particular theory, the XTEN of the CFXTEN compositions with the higher net charge are expected, as described above, to have less non-specific interactions with various negatively-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance. Conversely, the XTEN of the CFXTEN compositions with a low (or no) net charge are expected to have a higher degree of interaction with surfaces that, while contributing to active clearance, can potentiate the activity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850-9858). Thus, the invention provides CFXTEN in which the degree of potency, bioavailability, and half-life can be tailored by the selection and placement of the type and length of the XTEN in the CFXTEN compositions. Accordingly, the invention contemplates compositions in which a CF from Table 1 or from Table 2 and XTEN from Table 4 are substituted for the respective components of the foregoing examples, and are produced, for example, in a configuration from Table 6 or from formulas I-VII such that the construct has reduced clearance compared to an alternative configuration of the respective components. In some embodiments, the foregoing method for increasing the terminal half-life provides configured CFXTEN that can result in an increase in the terminal half-life of at least about 30%, or about 50%, or about 75%, or about 100%, or about 150%, or about 200%, or about 300%, or about 400% or more compared to the half-life of a CFXTEN in a second configuration where active clearance is not reduced. The invention further takes advantage of the fact that certain ligands wherein reduced binding to a clearance receptor, either as a result of a decreased on-rate or an increased off-rate, may be effected by the obstruction of either the N- or C-terminus and using that terminus as the linkage to another polypeptide of the composition, whether another molecule of a CF, an XTEN, or a spacer sequence results in the reduced binding. The choice of the particular configuration of the CFXTEN fusion protein reduces the degree of binding to a clearance receptor such that a reduced rate of active clearance is achieved.
• In cases where a reduction in active clearance is desired but retention of at least a portion of the biological activity is also desired, the CFXTEN is designed to retain sufficient biologic activity for the intact molecule. Thus, in one embodiment, the invention provides a CFXTEN configured such that the biologic activity of the CFXTEN is in the range of about 0.01%-40%, or about 0.01%-30%, or about 0.01%-20%, or about 0.01%-10 of the biological activity compared to the corresponding native coagulation factor. The biological activity of the configured CFXTEN is thus reduced by at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 99.99% as compared to the biological activity of the corresponding native coagulation factor not linked to XTEN, determined under comparable conditions. In the foregoing embodiments, the biological activity of the configured CFXTEN for the target receptor is “substantially reduced” compared to a corresponding native CF not linked to XTEN. Accordingly, the present invention provides compositions and methods to produce compositions with reduced biological activity but increased half-life by configuring the CFXTEN, examples of which are provided above, so as to be able to provide a desired in vivo biological response yet avoid active clearance mechanisms. The increased half-life permits higher dosages and reduced frequency of dosing compared to CF not linked to XTEN or compared to CFXTEN configurations wherein the fusion protein is subject to coagulation factor clearance mechanisms.
VI). Uses of the Compositions of the Present Invention
• In another aspect, the invention provides a method for achieving a beneficial effect in bleeding disorders and/or in a coagulation factor-related disease, disorder or condition mediated by FIX or FVII. As used herein, “coagulation factor-related diseases, disorders or conditions” is intended to include, but is not limited to bleeding disorders (e.g., defective platelet function, thrombocytopenia or von Willebrand's disease), coagulopathies (any disorder of blood coagulation, including coagulation factor deficiencies), hemophilia B (aka Christmas disease), factor IX-related bleeding disorders, factor VII deficiency, hemophilia A, vascular injury, uncontrolled bleeding in subjects not suffering from hemophilia, bleeding from trauma or surgery, bleeding due to anticoagulant therapy, and bleeding due to liver disease or conditions that can be ameliorated or corrected by administration of FIX or FVII to a subject. The present invention addresses disadvantages and/or limitations of other methods of treatment using factor IX or factor VII preparations that have a relatively short terminal half-life and/or a narrow therapeutic window.
• In some embodiments, the invention provides methods for treating a subject, such as a human, with a coagulation factor-related disease, disorder or condition comprising the step of administering to the subject a therapeutically- or prophylactically-effective amount of an CFXTEN wherein said administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the coagulation factor-related disease, disorder or condition. In one embodiment of the foregoing, the CFXTEN comprises a FVII. In another embodiment of the foregoing, the CFXTEN comprises a FIX. The effective amount produces a beneficial effect in helping to treat (e.g., cure or reduce the severity) or prevent (e.g., reduce the likelihood of onset or severity) a coagulation factor-related disease, disorder or condition. As used herein, “treating” means administering a drug or a biologic (e.g., a CFXTEN) to achieve an improvement in an existing disease, disorder or condition or preventing the occurrence of a disease, disorder or condition (including prophylaxis). A therapeutically-effective amount of a CFXTEN fusion protein can be that amount of composition that, when administered as a single or as repeated doses to a subject, leads to improvements in or amelioration of the underlying disease, disorder or condition, or improvements in signs or symptoms or physiologic parameters associated with the underlying disease, disorder or condition.
• Hemostasis is regulated by multiple protein factors, and such proteins, as well as analogues thereof, have found utility in the treatment of coagulation factor-related diseases, disorders and conditions. However, the use of commercially-available coagulation factors has met with less than optimal success in the management of subjects afflicted with such diseases, disorders and conditions. In particular, dose optimization and frequency of dosing is important for coagulation factors used in the treatment or prevention of bleeding episodes in coagulation factor-related diseases, disorders, or conditions, or uncontrolled bleeding in subjects not suffering from hemophilia. The fact that coagulation factors have a short half-life necessitates frequent dosing in order to achieve clinical benefit, which results in difficulties in the management of such patients.
• The invention provides methods of treatment comprising administering a CFXTEN composition to a subject suffering from or at risk of developing a coagulation factor-related disease, disorder or condition, wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition. In one embodiment, the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from hemophilia A wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition. In another embodiment, the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from hemophilia B wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition. In another embodiment, the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from factor VII deficiency wherein said administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition. In another embodiment, the method of treatment comprises administering a therapeutically-effective amount of an CFXTEN composition to a subject suffering from or at risk of developing uncontrolled bleeding wherein the administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with the condition. In most instances, the embodiments of the disclosed method of treatments utilizing a CFXTEN comprising a FVII are compositions in which the FVII has been activated; i.e., FVIIa. However, the invention also contemplates CFXTEN compositions in which the FVII has not been activated. Because of the comparatively long-half life of CFXTEN comprising FVII, it is believed that compositions comprising the inactive form of FVII that can be activated by mammalian endogenous proteases (because they include one or more cleavage sequences; e.g., the sequences of Table 7) or the fusion protein undergoes autoactivation such that 1) a bolus quantity of activated form of FVII is available by activation via clotting proteins of the intrinsic coagulation cascade that has been initiated; or 2) a persistent quantity of activated form of FVII is available by activation via proteases that are persistently or transiently present in the circulation; e.g., MMP-12, MMP-17, etc.
• Thus, the invention provides a method of treatment for a subject with a coagulation factor-related disease, disorder or conditions comprising administration of a CFXTEN comprising a FVII variant (as described above) wherein the FVII is not activated but has one or more cleavage sequences that, when cleaved by an endogenous protease, converts the FVII component to the activated form. In one embodiment of the foregoing, the method utilizes a CFXTEN composition that has a terminal half-life of at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 48 h, or at least about 96 h, or at least about 144 h, or at least about 160 h. Accordingly, the method represents a means to treat subjects with certain forms of chronic coagulopathies with what is essentially a “prodrug” form of FVII.
• In some embodiments, administration of the CFXTEN to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding CF component not linked to XTEN, determined using the same assay or based on a measured clinical parameter. In other embodiments, administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in activity in one or more of the biochemical, physiologic, or clinical parameters that is of longer duration than the activity of the corresponding CF component not linked to XTEN, determined using that same assay or based on a measured clinical parameter. In one embodiment, the administration of a therapeutically effective amount of a CFXTEN comprising a FVII to a subject results in a reduction in prothrombin time at about 2-7 days after administration of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject compared to the prothrombin time in a subject at a comparable time after administration of a comparable amount of FVII not linked to XTEN. In another embodiment, the administration of a CFXTEN comprising a FVII to a subject using a therapeutically effective amount results in maintenance of prothrombin times within 30% of normal in the subject for a period of time that is at least two-fold, or about three-fold, or at least about four-fold longer compared to a comparable dose regimen of FVII not linked to XTEN administered to a subject. In another embodiment, the administration of a therapeutically effective amount of a CFXTEN comprising a FIX to a subject results in a reduction in the activated partial prothrombin time at about 2-7 days after administration of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in the subject compared to the activated partial prothrombin time in a subject at a comparable time after administration of a comparable amount of FIX not linked to XTEN. In another embodiment, the administration of a CFXTEN comprising a FIX to a subject using a therapeutically effective amount results in maintenance of activated partial prothrombin times within 30% of normal in the subject for a period of time that is at least two-fold, or at least about three-fold, or at least about four-fold longer compared to a comparable dose regimen of FIX not linked to XTEN administered to a subject. In another embodiment, the administration of a CFXTEN comprising a FVII to a subject using a therapeutically effective amount results in maintenance of a bleeding time (in a bleeding time assay) within 30% of normal in the subject for a period of time that is at least two-fold, or about three-fold, or at least about four-fold longer compared to a comparable amount of FVII not linked to XTEN administered to a subject. In another embodiment, the administration of a CFXTEN comprising a FIX to a subject using a therapeutically effective amount results in maintenance of a bleeding time (in a bleeding time assay) within 30% of normal in the subject for a period of time that is at least two-fold, or about three-fold, or at least about four-fold longer compared to a comparable amount of FIX not linked to XTEN administered to a subject.
• As a result of the enhanced PK parameters of CFXTEN, as described herein, the CF is administered using longer intervals between doses compared to the corresponding CF not linked to XTEN to prevent, treat, alleviate, reverse or ameliorate symptoms or clinical abnormalities of the coagulation factor-related disease, disorder or condition or prolong the survival of the subject being treated. In a particular application, CFXTEN comprising FVII have utility in the treatment of hemophilia A and hemophilia B.
• It has been observed that FVIIa administered in high concentrations can function as a bypassing agent resulting in the activation of FX even in the absence of FIX or FVIII. In order to act as a bypassing agent FVIIa has to be dosed at concentrations that exceed the level of FVIIa in healthy people by approximately 100-fold. These levels are generally safe because FVIIa has low activity in the absence of tissue factor (TF), to which FVII binds. Tissue factor is released or presented on injured tissues which triggers clotting via the extrinsic system. The circulation half-life of FVIIa is in part limited by its inactivation by antithrombin (AT). Antithrombin can not bind to FVII but only to FVIIa. Thus, in one embodiment, the invention provides a method of treating hemophilia A or B by administering an amount of CFXTEN comprising an activated form of FVII, wherein the ability to activate FX in the circulation of a subject is maintained for a period that is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about 10-fold, or at least about 20-fold longer compared to FVII not linked to XTEN and administered to a comparable subject at a comparable dose. The current invention further provides CFXTEN fusion proteins comprising FVII linked to XTEN that can not be inactivated by AT by more than about 5% prior to its activation to FVIIa-XTEN. In one embodiment, the invention provides a method of treatment comprising administering a CFXTEN with a FVII component that is not activated, wherein the CFXTEN serves as a circulating depot wherein the area under the curve for the FVII that is activated to FVIIa and not complexed with AT is at least about two-fold greater, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about 10-fold, or at least about 20-fold greater than a FVII not linked to XTEN and administered at a comparable dose.
• In some embodiments of the method of treatment, (i) a smaller molar amount of (e.g. of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold-less or greater) the fusion protein is administered in comparison to the corresponding CF not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN; (ii) the fusion protein is administered less frequently (e.g., every two days, about every seven days, about every 14 days, about every 21 days, or about, monthly) in comparison to the corresponding CF not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN; or (iii) an accumulative smaller molar amount (e.g. about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less) of the fusion protein is administered in comparison to the corresponding CF not linked to the XTEN under the otherwise same dose regimen the fusion protein achieves a comparable therapeutic effect as the corresponding CF not linked to the XTEN. The accumulative smaller molar amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month. The therapeutic effect can be determined by any of the measured parameters or clinical endpoints described herein.
• The methods of the invention includes administration of consecutive doses of a therapeutically effective amount of the CFXTEN for a period of time sufficient to achieve and/or maintain the desired parameter or clinical effect, and such consecutive doses of a therapeutically effective amount establishes the therapeutically effective dose regimen for the CFXTEN, i.e., the schedule for consecutively administered doses of the fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in a sustained beneficial effect on any clinical sign or symptom, aspect, measured parameter or characteristic of a coagulation factor-related disease state or condition, including, but not limited to, those described herein. In one embodiment, the method comprises administering a therapeutically-effective amount of a pharmaceutical composition comprising a CFXTEN fusion protein composition comprising a CF linked to an XTEN sequence(s) and at least one pharmaceutically acceptable carrier to a subject in need thereof that results in greater improvement in at least one parameter, physiologic condition, or clinical outcome mediated by the CF component(s) (non-limiting examples of which are described above) compared to the effect mediated by administration of a pharmaceutical composition comprising a CF not linked to XTEN and administered at a comparable dose. In one embodiment, the pharmaceutical composition is administered at a therapeutically effective dose. In another embodiment, the pharmaceutical composition is administered using multiple consecutive doses using a therapeutically effective dose regimen (as defined herein) for the length of the dosing period.
• A therapeutically effective amount of the CFXTEN varies according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the administered fusion protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the CFXTEN are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount of CFXTEN required for the period of time necessary to achieve the desired prophylactic result; e.g., delayed onset of a bleeding episode. In the methods of treatment, the dose of the CFXTEN that is administered to a subject ranges from about 0.5 mg to 1000 mg/dose, or from about 1 mg to 400 mg/dose, or from about 10 mg to about 300 mg/dose for a 70 kg subject as loading and maintenance doses, depending on the weight of the subject and the severity of the condition.
• The method of treatment comprises administration of a CFXTEN using a therapeutically effective dose regimen to effect improvements in one or more parameters associated with coagulation factor diseases, disorders or conditions. In some embodiments, administration of the CFXTEN to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding CF component not linked to XTEN, determined using the same assay or based on a measured clinical parameter. In other embodiments, administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in activity in one or more of the biochemical, physiologic, or clinical parameters that is of longer duration than the activity of one of the single CF components not linked to XTEN, determined using that same assay or based on a measured clinical parameter. In one embodiment of the foregoing, the administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in an improvement in prothrombin time or activated partial thromboplastin time of at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100% or more in the subject compared to a comparable dose of CF not linked to XTEN administered to a subject. In another embodiment of the foregoing, the administration of the CFXTEN to a subject using a therapeutically effective dose regimen results in decreased instances of bleeding in the subject of at least about 10%, or about 20%, or about 30%, or about 40%, or about 50% or more compared to a comparable dose regimen of CF not linked to XTEN administered to a subject.
• The invention further contemplates that CFXTEN used in accordance with the methods provided herein is administered in conjunction with other treatment methods and compositions (e.g., other coagulation proteins) useful for treating coagulation factor-related diseases, disorders, and conditions, or conditions for which coagulation factor is adjunctive therapy; e.g., bleeding episodes due to injury or surgery.
• In another aspect, the invention provides a method of designing the CFXTEN compositions with desired pharmacologic or pharmaceutical properties. The CFXTEN fusion proteins are designed and prepared with various objectives in mind (compared to the CF components not linked to the fusion protein), including improving the therapeutic efficacy for the treatment of coagulation factor-related diseases, disorders, and conditions, enhancing the pharmacokinetic characteristics of the fusion proteins compared to the CF, lowering the dose or frequency of dosing required to achieve a pharmacologic effect, enhancing the pharmaceutical properties, and to enhance the ability of the CF components to remain within the therapeutic window for an extended period of time.
• In general, the steps in the design and production of the fusion proteins and the inventive compositions, as illustrated inFIGS. 31-33, include: (1) the selection of CFs (e.g., native proteins, sequences of Tables 1 and 2, analogs or derivatives with activity) to treat the particular disease, disorder or condition; (2) selecting the XTEN that will confer the desired PK and physicochemical characteristics on the resulting CFXTEN (e.g., the administration of the CFXTEN composition to a subject results in the fusion protein being maintained within the therapeutic window for a greater period compared to CF not linked to XTEN); (3) establishing a desired N- to C-terminus configuration of the CFXTEN to achieve the desired efficacy or PK parameters; (4) establishing the design of the expression vector encoding the configured CFXTEN; (5) transforming a suitable host with the expression vector; and (6) expression and recovery of the resultant fusion protein. For those CFXTEN for which an increase in half-life (greater than 24 h) or an increased period of time spent within a therapeutic window is desired, the XTEN chosen for incorporation generally has at least about 100, or about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues where a single XTEN is to be incorporated into the CFXTEN. In another embodiment, the CFXTEN comprises a first XTEN of the foregoing lengths, and at least a second XTEN of about 36, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues.
• In other embodiments, where an increase in half-life is not required, but an increase in a pharmaceutical property (e.g., solubility) is desired, a CFXTEN is designed to include XTEN of shorter lengths. In some embodiments of the foregoing, the CFXTEN comprises a CF linked to an XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96 amino acid residues, in which the solubility of the fusion protein under physiologic conditions is at least three-fold greater than the corresponding CF not linked to XTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 60-fold or greater than CF not linked to XTEN. In one embodiment of the foregoing, the CF is factor IX. In another embodiment, the CF is factor VII. In another embodiment, the XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from Tables 4, and 9-13.
• In another aspect, the invention provides methods of making CFXTEN compositions to improve ease of manufacture, result in increased stability, increased water solubility, and/or ease of formulation, as compared to the native CF. In one embodiment, the invention includes a method of increasing the water solubility of a CF comprising the step of linking the CF to one or more XTEN such that a higher concentration in soluble form of the resulting CFXTEN can be achieved, under physiologic conditions, compared to the CF in an un-fused state. Factors that contribute to the property of XTEN to confer increased water solubility of CFs when incorporated into a fusion protein include the high solubility of the XTEN fusion partner and the low degree of self-aggregation between molecules of XTEN in solution. In some embodiments, the method results in a CFXTEN fusion protein wherein the water solubility is at least about 20%, or at least about 30% greater, or at least about 50% greater, or at least about 75% greater, or at least about 90% greater, or at least about 100% greater, or at least about 150% greater, or at least about 200% greater, or at least about 400% greater, or at least about 600% greater, or at least about 800% greater, or at least about 1000% greater, or at least about 2000% greater, or at least about 4000% greater, or at least about 6000% greater under physiologic conditions, compared to the un-fused CF. In one embodiment, the XTEN of the CFXTEN fusion protein is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from Tables 4, and 9-13.
• In another embodiment, the invention includes a method of increasing the shelf-life of a CF comprising the step of linking the CF with one or more XTEN selected such that the shelf-life of the resulting CFXTEN is extended compared to the CF in an un-fused state. As used herein, shelf-life refers to the period of time over which the functional activity of a CF or CFXTEN that is in solution or in some other storage formulation remains stable without undue loss of activity. As used herein, “functional activity” refers to a pharmacologic effect or biological activity, such as the ability to bind a receptor or ligand, or substrate, or an enzymatic activity, or to display one or more known functional activities associated with a CF, as known in the art. A CF that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to one that remains in solution. Factors that contribute to the ability of the method to extend the shelf life of CFs when incorporated into a fusion protein include increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion partner. In particular, the low tendency of XTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of CFs, and the heat-stability of XTEN contributes to the property of CFXTEN fusion proteins to remain soluble and functionally active for extended periods. In one embodiment, the method results in CFXTEN fusion proteins with “prolonged” or “extended” shelf-life that exhibit greater activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the un-fused full-length CF. In one embodiment, the method includes the step of formulating the isolated CFXTEN with one or more pharmaceutically acceptable excipients that enhance the ability of the XTEN to retain its unstructured conformation and for the CFXTEN to remain soluble in the formulation for a time that is greater than that of the corresponding un-fused CF. In one embodiment, the method comprises linking a CF to one or more XTEN selected from Tables 4 and 9-13 to create a CFXTEN fusion protein results in a solution that retains greater than about 100% of the functional activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the functional activity of a standard when compared at a given time point and when subjected to the same storage and handling conditions as the standard, thereby increasing its shelf-life.
• Shelf-life may also be assessed in terms of functional activity remaining after storage, normalized to functional activity when storage began. CFXTEN fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended functional activity retain about 50% more functional activity, or about 60%, 70%, 80%, or 90% more of the functional activity of the equivalent CF not linked to XTEN when subjected to the same conditions for the same period of time. For example, a CFXTEN fusion protein of the invention comprising coagulation factor fused to one or more XTEN sequences selected from Tables 4 and 9-13 retains about 80% or more of its original activity in solution for periods of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions. In some embodiments, the CFXTEN retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and most preferably at least about 90% or more of its original activity in solution when heated at 80° C. for 10 min. In other embodiments, the CFXTEN retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or alternatively at least about 90% or more of its original activity in solution when heated or maintained at 37° C. for about 7 days. In another embodiment, CFXTEN fusion protein retains at least about 80% or more of its functional activity after exposure to a temperature of about 30° C. to about 70° C. over a period of time of about one hour to about 18 hours. In the foregoing embodiments hereinabove described in this paragraph, the retained activity of the CFXTEN is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of the corresponding CF not linked to the fusion protein.
VII). The Nucleic Acids Sequences of the Invention
• The present invention provides isolated polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion proteins, including homologous variants thereof. In another aspect, the invention encompasses methods to produce polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion protein, including homologous variants thereof. In general, and as illustrated inFIGS. 4-6, the methods of producing a polynucleotide sequence coding for a CFXTEN fusion protein and expressing the resulting gene product include assembling nucleotides encoding CF and XTEN, ligating the components in frame, incorporating the encoding gene into an expression vector appropriate for a host cell, transforming the appropriate host cell with the expression vector, and culturing the host cell under conditions causing or permitting the fusion protein to be expressed in the transformed host cell, thereby producing the biologically-active CFXTEN polypeptide, which is recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology is used to make the polynucleotides and expression vectors of the present invention.
• In accordance with the invention, nucleic acid sequences that encode CFXTEN (or its complement) is used to generate recombinant DNA molecules that direct the expression of CFXTEN fusion proteins in appropriate host cells. Several cloning strategies are suitable for performing the present invention, many of which is used to generate a construct that comprises a gene coding for a fusion protein of the CFXTEN composition of the present invention, or its complement. In some embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises at least a first CF and at least a first XTEN polypeptide, or their complement. In one embodiment of the foregoing, the gene comprises a sequence encoding a CF or sequence variant. In other embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises nucleotides encoding at least a first molecule of CF or its complement and a first and at least a second XTEN or their complement that is used to transform a host cell for expression of the fusion protein of the CFXTEN composition. In the foregoing embodiments hereinabove described in this paragraph, the genes can further comprise nucleotides encoding spacer sequences that also encode cleavage sequence(s).
• In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of the XTEN of the inventive compositions is achieved despite use of a “building block” molecular approach in the creation of the XTEN-encoding sequences. This was achieved by the use of a library of polynucleotides encoding peptide sequence motifs, described above, that are then ligated and/or multimerized to create the genes encoding the XTEN sequences (seeFIGS. 4 and 5 and Examples). Thus, while the XTEN(s) of the expressed fusion protein may consist of multiple units of as few as four different sequence motifs, because the motifs themselves consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered non-repetitive. Accordingly, in one embodiment, the XTEN-encoding polynucleotides comprise multiple polynucleotides that encode non-repetitive sequences, or motifs, operably linked in frame and in which the resulting expressed XTEN amino acid sequences are non-repetitive.
• In one approach, a construct is first prepared containing the DNA sequence corresponding to CFXTEN fusion protein. DNA encoding the CF of the compositions is obtained from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess CF mRNA and to express it at a detectable level. Libraries is screened with probes containing, for example, about 20 to 100 bases designed to identify the CF gene of interest by hybridization using conventional molecular biology techniques. The best candidates for probes are those that represent sequences that are highly homologous for coagulation factor, and should be of sufficient length and sufficiently unambiguous that false positives are minimized, but may be degenerate at one or more positions. If necessary, the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. One can then use polymerase chain reaction (PCR) methodology to amplify the target DNA or RNA coding sequence to obtain sufficient material for the preparation of the CFXTEN constructs containing the CF gene(s). Assays can then be conducted to confirm that hybridizing full-length genes are the desired CF gene(s). By these conventional methods, DNA can be conveniently obtained from a cDNA library prepared from such sources. The CF encoding gene(s) is also be obtained from a genomic library or created by standard synthetic procedures known in the art (e.g., automated nucleic acid synthesis using, for example one of the methods described in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA sequences obtained from publicly available databases, patents, or literature references. Such procedures are well known in the art and well described in the scientific and patent literature. For example, sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or GenBank Accession Numbers (e.g., Locus ID, NP_XXXXX, and XP_XXXXX) Model Protein identifiers available through the National Center for Biotechnology Information (NCBI) webpage, available on the world wide web at ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid sequence of the protein of interest or of a fragment or variant of the protein. For such sequence identifiers provided herein, the summary pages associated with each of these CAS and GenBank and GenSeq Accession Numbers as well as the cited journal publications (e.g., PubMed ID number (PMID)) are each incorporated by reference in their entireties, particularly with respect to the amino acid sequences described therein. In one embodiment, the CF encoding gene encodes a protein from any one of Table 1 or Table 2, or a fragment or variant thereof.
• A gene or polynucleotide encoding the CF portion of the subject CFXTEN protein, in the case of an expressed fusion protein that comprises a single CF is then be cloned into a construct, which is a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system. In a later step, a second gene or polynucleotide coding for the XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the CF gene by cloning it into the construct adjacent and in frame with the gene(s) coding for the CF. This second step occurs through a ligation or multimerization step. In the foregoing embodiments hereinabove described in this paragraph, it is to be understood that the gene constructs that are created can alternatively be the complement of the respective genes that encode the respective fusion proteins.
• The gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longer XTEN genes of a desired length and sequence. In one embodiment, the method ligates two or more codon-optimized oligonucleotides encoding XTEN motif or segment sequences of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 36 amino acids, or about 48 to about 144 amino acids, or about 144 to about 288 or longer, or any combination of the foregoing ranges of motif or segment lengths.
• Alternatively, the disclosed method is used to multimerize XTEN-encoding sequences into longer sequences of a desired length; e.g., a gene encoding 36 amino acids of XTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, etc. Even with multimerization, XTEN polypeptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitiveness through design of the codons selected for the motifs of the shortest unit being used, which can reduce recombination and increase stability of the encoding gene in the transformed host. Genes encoding XTEN with non-repetitive sequences is assembled from oligonucleotides using standard techniques of gene synthesis. The gene design can be performed using algorithms that optimize codon usage and amino acid composition. In one method of the invention, a library of relatively short XTEN-encoding polynucleotide constructs is created and then assembled, as illustrated inFIGS. 4 and 5. This can be a pure codon library such that each library member has the same amino acid sequence but many different coding sequences are possible. Such libraries can be assembled from partially randomized oligonucleotides and used to generate large libraries of XTEN segments comprising the sequence motifs. The randomization scheme can be optimized to control amino acid choices for each position as well as codon usage. Exemplary methods to achieve the foregoing are disclosed in the Examples.
Polynucleotide Libraries
• In another aspect, the invention provides libraries of polynucleotides that encode XTEN sequences that are used to assemble genes that encode XTEN of a desired length and sequence.
• In certain embodiments, the XTEN-encoding library constructs comprise polynucleotides that encode polypeptide segments of a fixed length. As an initial step, a library of oligonucleotides that encode motifs of 9-14 amino acid residues can be assembled. In a preferred embodiment, libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.
• The XTEN-encoding sequence segments can be dimerized or multimerized into longer encoding sequences. Dimerization or multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. This process of can be repeated multiple times until the resulting XTEN-encoding sequences have reached the organization of sequence and desired length, providing the XTEN-encoding genes. As will be appreciated, a library of polynucleotides that encodes, e.g., 12 amino acid motifs can be dimerized and/or ligated into a library of polynucleotides that encode 36 amino acids. Libraries encoding motifs of different lengths; e.g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids are contemplated by the invention. In turn, the library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the Examples) can be serially dimerized into a library containing successively longer lengths of polynucleotides that encode XTEN sequences of a desired length for incorporation into the gene encoding the CFXTEN fusion protein, as disclosed herein. In some embodiments, libraries are assembled of polynucleotides that encode amino acids that are limited to specific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. In other embodiments, libraries comprise sequences that encode two or more of the motif family sequences from Table 3. The names and sequences of representative, non-limiting polynucleotide sequences of libraries that encode 36mers are presented in Tables 9-12, and the methods used to create them are described more fully in the respective Examples. In other embodiments, libraries that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of the codons are selected from the group consisting of condons for glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) amino acids. The libraries can be used, in turn, for serial dimerization or ligation to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths, in which the encoded XTEN can have one or more of the properties disclosed herein, when expressed as a component of a CFXTEN fusion protein. In some cases, the polynucleotide library sequences may also include additional bases used as “sequencing islands,” described more fully below.
• FIG. 5 is a schematic flowchart of representative, non-limiting steps in the assembly of a XTEN polynucleotide construct and a CFXTEN polynucleotide construct in the embodiments of the invention.Individual oligonucleotides501 are annealed intosequence motifs502 such as a 12 amino acid motif (“12-mer”), which is subsequently ligated with an oligo containing BbsI, andKpnI restriction sites503. Additional sequence motifs from a library are annealed to the 12-mer until the desired length of theXTEN gene504 is achieved. The XTEN gene is cloned into a stuffer vector. The vector optionally encodes aFlag sequence506 followed by a stuffer sequence that is flanked by BsaI, BbsI, andKpnI sites507 and, in this case, a single CF gene (encoding FIX in this example)508, resulting in the gene encoding a CFXTEN comprising asingle CF500. A non-exhaustive list of the XTEN names for polynucleotides encoding XTEN and precursor sequences is provided in Table 8.

• TABLE 8
  DNA sequences of XTEN and precursor sequences

  	SEQ
  XTEN	ID
  Name	NO:	DNA Nucleotide Sequence
  AE48	111	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGC
  		GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTC
  		CAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCT
  AM48	112	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGC
  		ACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTC
  		CAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCT
  AE144	113	GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCT
  		ACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA
  		GGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAAGGTACCTCTACTGAACCT
  		TCTGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA
  		GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCAGGTAGCGAACCGGCTACT
  		TCCGGTTCTGAAACTCCAGGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA
  		GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT
  		TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA
  AF144	114	GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTG
  		AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGG
  		TTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTACCAGCGAATCCCCG
  		TCTGGCACCGCACCAGGTTCTACTAGCTCTACCGCAGAATCTCCGGGTCCAGGT
  		ACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCGGAAAGCG
  		GCTCCGCATCTCCAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCAGGTAC
  		CTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCT
  		TCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACCA
  AE288	115	GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACC
  		TCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA
  		GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCT
  		ACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA
  		GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCA
  		ACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA
  		GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCT
  		CCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA
  		GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCT
  		ACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA
  		GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT
  		TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA
  		GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT
  		TCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA
  		GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCT
  		ACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA
  AE576	116	GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCT
  		ACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA
  		GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT
  		TCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA
  		GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT
  		TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA
  		GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA
  		ACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA
  		GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCT
  		CCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA
  		GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCT
  		ACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA
  		GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT
  		TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA
  		GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCA
  		ACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCC
  		AGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGC
  		AACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC
  		AGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACC
  		GTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC
  		AGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACC
  		TTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACC
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTC
  		TCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACC
  		AGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  		CTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCC
  		AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACC
  		AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC
  		TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA
  		AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGC
  		AACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA
  AF576	117	GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCCACTAGCTCTACCG
  		CAGAATCTCCGGGCCCAGGTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCAG
  		GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGC
  		AGAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGT
  		TCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAAT
  		CTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTC
  		TACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCT
  		TCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTA
  		CCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCGAATCTTC
  		TACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACT
  		AGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGC
  		ACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTA
  		GCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTACCCCTGAAAGCGGCTCCGC
  		TTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCCGGGTCCAGGTTCTACTAGC
  		TCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCA
  		TCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCG
  		AATCCCCGTCTGGTACCGCACCAGGTACTTCTACCCCGGAAAGCGGCTCTGCTT
  		CTCCAGGTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCAGGTTCTACTAGCG
  		AATCTCCTTCTGGTACCGCTCCAGGTTCTACCAGCGAATCCCCGTCTGGTACTGC
  		TCCAGGTTCTACCAGCGAATCTCCTTCTGGTACTGCACCAGGTTCTACTAGCTCT
  		ACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCT
  		CCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAGCGAAT
  		CTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACC
  		AGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACTAGCGAATCT
  		CCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCA
  		GGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTACTTCTCCGAGCGGTG
  		AATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGG
  		TACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTTCCACTAGCTCTACTGCT
  		GAATCTCCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGTT
  		CTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTTCTACTAGCTCTACTGCAG
  		AATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTA
  		CTTCTACCCCTGAAAGCGGTTCTGCATCTCCA
  AE624	118	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGC
  		GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTC
  		CAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCT
  		CTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCC
  		AGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTC
  		TCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACC
  		AGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGC
  		TACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCC
  		AGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTC
  		TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCC
  		AGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGA
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACC
  		TTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCC
  		AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC
  		AACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCC
  		AGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACC
  		GTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCC
  		AGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTC
  		TCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC
  		AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCC
  		AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC
  		AGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGA
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGC
  		AACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC
  		AGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC
  		CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC
  		AGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC
  		TACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGA
  		AGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTC
  		TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCC
  		AGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA
  AM875	119	GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACT
  		TCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA
  		GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAA
  		AGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG
  		GTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAG
  		CGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGT
  		AGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT
  		CCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGT
  		ACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC
  		CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT
  		ACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCT
  		ACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT
  		ACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACC
  		CCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT
  		ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCC
  		GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGT
  		ACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCT
  		GGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGT
  		AGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG
  		CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTAC
  		CTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGA
  		GGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAG
  		CCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT
  		TCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCA
  		AGCGCAAGCGGCGCGCCAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGA
  		GTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
  		GGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCT
  		CCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCC
  		CTAGCGGTGAATCTTCTACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTC
  		CTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCG
  		TCTGCATCTACCGGTACCGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAA
  		ACTCCAGGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCG
  		GCTACTTCCGGCTCTGAAACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCG
  		GGCCCAGGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTA
  		GCGGCGAATCTTCTACCGCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAA
  		CTCCAGGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTG
  		AACCTTCTGAGGGCAGCGCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTG
  		GTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCG
  		AATCTCCTTCTGGCACTGCACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCG
  		CTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCG
  		AACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCT
  		CCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGG
  		CACCAGCTCTACTGGTTCTCCAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACC
  		CCAGGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGT
  		TCTCCTACCTCCACTGAGGAAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCC
  		CAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCAC
  		CAGCTCTACTGGTTCTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCC
  		AGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACC
  		GTCCGAAGGTAGCGCACCA
  AE864	120	GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCT
  		ACTCCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA
  		GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCT
  		TCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA
  		GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACT
  		TCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA
  		GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCA
  		ACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA
  		GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCT
  		CCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA
  		GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCT
  		ACCCCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA
  		GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACT
  		TCTGGCTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA
  		GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCA
  		ACCCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA
  		AGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGC
  		AACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC
  		AGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACC
  		GTCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACC
  		AGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACC
  		TTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACC
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTC
  		TCCTACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACC
  		AGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  		CTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCC
  		AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACC
  		AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC
  		TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA
  		AGGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGC
  		AACCCCGGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACC
  		AGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  		CTCCGGCTCTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCC
  		AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACC
  		AGGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGC
  		AACCCCTGAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCC
  		AGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTC
  		TCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGA
  		AGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC
  		TACCCCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCC
  		AGGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTAC
  		TTCTGGTTCTGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCC
  		AGGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACTTCTACTGAACC
  		TTCCGAAGGCAGCGCACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCC
  		AGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA
  AF864	121	GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTACCTCTCCTAGCGGCG
  		AATCTTCTACCGCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGG
  		TTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGC
  		GGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTT
  		CTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTC
  		TGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCACCAGGTTCT
  		ACTAGCGAATCTCCGTCTGGCACTGCTCCAGGTACTTCTCCTAGCGGTGAATCTT
  		CTACCGCTCCAGGTACTTCCCCTAGCGGCGAATCTTCTACCGCTCCAGGTTCTAC
  		TAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCTCTCCTAGCGGTGAATCTTCT
  		ACCGCTCCAGGTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCAGGTTCTACTA
  		GCTCTACTGCAGAATCTCCTGGCCCAGGTACCTCTACTCCGGAAAGCGGCTCTG
  		CATCTCCAGGTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCAGGTTCTACTAG
  		CGAATCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACT
  		GCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCAGGTTCTACCAGCT
  		CTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCAT
  		CTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTACTTCTCCGAG
  		CGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAATCTCCGGGC
  		CCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTACTCCTG
  		AAAGCGGTTCTGCATCTCCAGGTTCCACTAGCTCTACCGCAGAATCTCCGGGCC
  		CAGGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGTTCTACTAGCTCTAC
  		TGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCA
  		GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGGTTCTACTAGCGAATCTC
  		CTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG
  		GTACCTCTACCCCTGAAAGCGGTCCXXXXXXXXXXXXTGCAAGCGCAAGCGGC
  		GCGCCAAGCACGGGAXXXXXXXXTAGCGAATCTCCTTCTGGTACCGCTCCAGGT
  		TCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTT
  		CTGGTACTGCACCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTTC
  		TACCAGCGAATCCCCGTCTGGTACTGCTCCAGGTTCTACCAGCGAATCTCCTTCT
  		GGTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACT
  		TCTCCTAGCGGTGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTT
  		CTACTGCTCCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTC
  		CCCGAGCGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTC
  		CGCTTCTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACT
  		AGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCT
  		ACCGCACCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCT
  		ACCCCGGAAAGCGGCTCTGCTTCTCCAGGTACTTCTACCCCGGAAAGCGGCTCC
  		GCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGTACTTCTA
  		CCCCTGAAAGCGGCTCCGCTTCTCCAGGTTCCACTAGCTCTACCGCTGAATCTCC
  		GGGTCCAGGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTACTAGC
  		GAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCG
  		CACCAGGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGTACTTCCCCGAG
  		CGGTGAATCTTCTACTGCACCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCT
  		CCAGGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGCG
  		GCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCACC
  		AGGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGTTCTACCAGCTCTACT
  		GCTGAATCTCCTGGTCCAGGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA
  		GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGTAGCTCTACTCCGTCTG
  		GTGCAACTGGCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA
  		XXXX was inserted in two areas where no sequence information is available.
  AG864	122	GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTT
  		CTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCAGG
  		TACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGT
  		GCTACCGGCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTG
  		CTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTACCCCGGGCAGCGGTACCG
  		CATCTTCTTCTCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGTAC
  		TCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCT
  		ACTGGTTCTCCAGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTACCC
  		CGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAAC
  		CGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCCCG
  		GGTAGCGGTACCGCTTCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCG
  		GCTCTCCAGGTTCTAACCCTTCTGCATCCACCGGTACCGGCCCAGGTTCTAGCCC
  		TTCTGCTTCCACCGGTACTGGCCCAGGTAGCTCTACCCCTTCTGGTGCTACCGGC
  		TCCCCAGGTAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCAGGTGCATCTCCGG
  		GCACTAGCTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTC
  		TCCAGGTGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTACTCCTGGCAGC
  		GGTACCGCTTCTTCTTCTCCAGGTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCC
  		AGGTGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCCCCGGGCACT
  		AGCTCTACCGGTTCTCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAG
  		GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTGCATCTCCGGGCACTAG
  		CTCTACTGGTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGT
  		GCTTCTCCTGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACTCCGTCTGGTG
  		CAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGC
  		ATCCCCTGGCACCAGCTCTACCGGTTCTCCAGGTACCCCGGGCAGCGGTACCGC
  		ATCTTCCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGC
  		TCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACTCCGTCTGGTGCAA
  		CCGGCTCCCCAGGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGGTGCTTC
  		CCCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCCCCGGGTACCAGCTCTAC
  		CGGTTCTCCAGGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGGTGCTTCT
  		CCGGGCACCAGCTCTACTGGTTCTCCAGGTGCATCTCCGGGCACTAGCTCTACTG
  		GTTCTCCAGGTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGTGCTTCTCC
  		TGGTACCAGCTCTACTGGTTCTCCAGGTACCCCTGGTAGCGGTACTGCTTCTTCC
  		TCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGTACCCCGGGTA
  		GCGGTACCGCATCTTCTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGTTC
  		TCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCT
  		TCTGGTGCTACTGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCC
  		CAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGTTCTAGCCCGTCTGC
  		ATCTACTGGTACTGGTCCAGGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCA
  		GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAGGTAGCTCTACTCCTTCTG
  		GTGCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGG
  		TTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCTCCGGGTACTAGC
  		TCTACTGGTTCTCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTA
  		GCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCCTTCTGCATCTAC
  		CGGTACTGGTCCAGGTGCATCCCCTGGTACCAGCTCTACCGGTTCTCCAGGTTCT
  		AGCCCTTCTGCTTCTACCGGTACCGGTCCAGGTACCCCTGGCAGCGGTACCGCA
  		TCTTCCTCTCCAGGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGTAGCT
  		CTACTCCTTCTGGTGCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTAC
  		CGGTTCTCCA
  AM923	123	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCATCCCCGGGC
  		ACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTC
  		CAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTACTTCTACTGAACC
  		GTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCC
  		AGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTAC
  		CGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAAAGCGGCTCTGCATCTCC
  		AGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGTTCTACTAGCGAATCC
  		CCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAG
  		GTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGTAGCGAACCGGCAACCT
  		CCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAG
  		GTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGTACCTCTACTGAACCTT
  		CTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAG
  		GTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACCGT
  		CCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAG
  		GTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACTTCTACCGAACCTT
  		CCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAG
  		GTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTTC
  		CGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAG
  		GTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTACTTCTACTGAACCTTC
  		CGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGG
  		TAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGTAGCTCTACCCCGTCTGG
  		TGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGT
  		AGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTACCTCTACCGAACCGTCCG
  		AGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTA
  		GCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAGCCCTGCTGGCTCTCCGA
  		CTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTA
  		CTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTGCAAGCGCAAGCGGCGCGC
  		CAAGCACGGGAGGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCAGGTAGCC
  		CGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCCGGCTGGCTCTCCAACTT
  		CTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGTTCTAC
  		TAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCT
  		ACTGCACCAGGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGTAGCTCTA
  		CCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAGCCCGTCTGCATCTACCGGTAC
  		CGGCCCAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACTTCTGA
  		AAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCGGCTACTTCCGGCTCTGA
  		AACCCCAGGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGGTTCTACTAG
  		CTCTACTGCAGAATCTCCGGGTCCAGGTACTTCTCCTAGCGGCGAATCTTCTACC
  		GCTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGCGAACCT
  		GCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACTGAACCTTCTGAGGGCAGC
  		GCACCAGGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTC
  		CGGAAAGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGC
  		ACCAGGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACCTCTACTGA
  		ACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGCGC
  		ACCAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCT
  		GCTTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTC
  		CAGGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCG
  		CAACTCCGGAGTCTGGTCCAGGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGG
  		AAGGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGC
  		TTCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA
  		GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCG
  		TCTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA
  AE912	124	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACCCCGGGTAGC
  		GGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTC
  		CAGGTGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCCCGGCTGGCT
  		CTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCC
  		AGGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCCCAGCAGGCTC
  		TCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACC
  		AGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGC
  		TACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCC
  		AGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCGGCAGGCTC
  		TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCC
  		AGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGA
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACC
  		TTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCC
  		AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGC
  		AACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCC
  		AGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACC
  		GTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCC
  		AGGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTAGCCCTGCTGGCTC
  		TCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC
  		AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACCTCTGAAAGCGC
  		TACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCC
  		AGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCC
  		AGGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACC
  		GTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGA
  		AGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTGAAAGCGC
  		AACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC
  		AGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC
  		CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC
  		AGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGC
  		TACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGA
  		AGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTAGCCCGGCAGGCTC
  		TCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCC
  		AGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTACCTCTGAAAGCGC
  		AACTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCC
  		AGGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCAGGTAGCGAACCTGCAAC
  		CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC
  		AGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCAGGTAGCCCTGCTGGCTC
  		TCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCC
  		AGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTACTTCTGAAAGCGC
  		TACTCCTGAGTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGA
  		AGGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTACCGAACC
  		TTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCC
  		AGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTGAAAGCGC
  		TACCCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCC
  		AGGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCAGGTAGCCCAGCAGGCTC
  		TCCGACTTCCACTGAGGAAGGTACTTCTACTGAACCTTCCGAAGGCAGCGCACC
  		AGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTAGCGAACCTGCAAC
  		CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCC
  		AGGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA
  AM1318	125	GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGGTAGCGAACCGGCTACT
  		TCCGGTTCTGAAACCCCAGGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA
  		GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGTACCTCTACTCCGGAA
  		AGCGGCTCTGCATCTCCAGGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG
  		GTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCTACTCCTGAAAG
  		CGGTTCCGCTTCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCTCCAGGT
  		AGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT
  		CCTGAATCCGGCCCAGGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAAGGT
  		ACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC
  		CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAGGT
  		ACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTAGCCCAGCAGGTTCTCCT
  		ACCTCCACCGAGGAAGGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGGT
  		ACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCTACC
  		CCTGAGTCCGGCCCAGGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGT
  		ACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCC
  		GAGGGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGT
  		ACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCT
  		GGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAAGGT
  		AGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG
  		CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCAGGTAC
  		CTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTGA
  		GGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCAGGTAG
  		CCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT
  		TCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTCCA
  		GAACCAACGGGGCCGGCCCCAAGCGGAGGTAGCGAACCGGCAACCTCCGGCTC
  		TGAAACCCCAGGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCC
  		GGCAGGTTCTCCGACTTCCACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGA
  		GTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
  		GGCTGGCTCTCCAACTTCTACTGAAGAAGGTACTTCTGAAAGCGCTACTCCTGA
  		GTCCGGCCCAGGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAAGGTAGCCC
  		GGCTGGCTCTCCAACTTCTACTGAAGAAGGTTCTACCAGCTCTACCGCTGAATCT
  		CCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGGTACTTCCC
  		CTAGCGGTGAATCTTCTACTGCACCAGGTTCTACCAGCGAATCTCCTTCTGGCAC
  		CGCTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCT
  		AGCGGCGAATCTTCTACCGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGC
  		GCACCAGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA
  		AGCGCTACTCCTGAATCCGGTCCAGGTAGCGAACCGGCAACCTCTGGCTCTGAA
  		ACCCCAGGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA
  		AGCGCTACTCCGGAATCCGGTCCAGGTACCTCTACTGAACCTTCTGAGGGCAGC
  		GCTCCAGGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT
  		GAACCGTCCGAAGGTAGCGCACCAGGTACCTCCCCTAGCGGCGAATCTTCTACT
  		GCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGTACCTCCCCTA
  		GCGGTGAATCTTCTACCGCACCAGGTACTTCTACCGAACCGTCCGAGGGTAGCG
  		CACCAGGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGGTACTTCTACCG
  		AACCGTCCGAGGGTAGCGCACCAGGTTCTAGCCCTTCTGCTTCCACCGGTACCG
  		GCCCAGGTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTAGCTCTACTCC
  		GTCTGGTGCAACCGGCTCCCCAGGTAGCTCTACCCCGTCTGGTGCTACCGGCTCT
  		CCAGGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGGTGCATCCCCGGGT
  		ACTAGCTCTACCGGTTCTCCAGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGG
  		AGGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACC
  		GCTGAATCTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCAG
  		GTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGT
  		CTGAGGGTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCAG
  		GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGG
  		TGCTACCGGCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT
  		ACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAAT
  		CTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCAGGTAC
  		TTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGG
  		CTCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCAGGTTC
  		TACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCT
  		GGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGC
  		CCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG
  		GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCAGGTAGC
  		CCTGCTGGCTCTCCAACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCT
  		GAATCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCAGGTAGC
  		TCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTA
  		CCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGTTCTAC
  		TAGCGAATCCCCGTCTGGTACTGCTCCAGGTACTTCCCCTAGCGGTGAATCTTCT
  		ACTGCTCCAGGTTCTACCAGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCT
  		ACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCATCCCCGGGTACCAGCTCTACC
  		GGTTCTCCAGGTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCAGGTAGCCCTG
  		CTGGCTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTAC
  		TGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA
  BC864	126	GGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGAACCA
  		TCCGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACCATCA
  		GGTAGCGGCGCATCCGAGCCTACCTCTACTGAACCAGGTAGCGAACCGGCTACC
  		TCCGGTACTGAGCCATCAGGTAGCGAACCGGCAACTTCCGGTACTGAACCATCA
  		GGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCTGAG
  		CCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGCGCA
  		GGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGAACCA
  		TCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCCATCA
  		GGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACCA
  		TCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGCA
  		GGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAGCAAC
  		TTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAGGTGC
  		AGGTAGCGGCGCATCCGAACCTACTTCCACTGAACCAGGTACTAGCGAGCCATC
  		CACCTCTGAACCAGGTGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATC
  		AGGTAGCGAACCGGCTACCTCTGGTACTGAACCATCAGGTACTTCTACCGAACC
  		ATCCGAGCCTGGTAGCGCAGGTACTTCTACCGAACCATCCGAGCCAGGCAGCGC
  		AGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGCAA
  		CTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACCAT
  		CAGGTAGCGAACCGGCTACTTCCGGCACTGAACCATCAGGTAGCGAACCAGCA
  		ACCTCCGGTACTGAACCATCAGGTACTTCCACTGAACCATCCGAACCGGGTAGC
  		GCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATCT
  		GAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAGC
  		GCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCATCT
  		GAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCAGC
  		GCAGGTAGCGGCGCATCTGAACCAACCTCTACTGAACCAGGTAGCGAACCAGC
  		AACTTCTGGTACTGAACCATCAGGTAGCGGCGCATCTGAGCCTACTTCCACTGA
  		ACCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATC
  		TGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAG
  		CGCAGGTAGCGAACCGGCAACTTCCGGCACTGAACCATCAGGTAGCGGTGCATC
  		TGAGCCGACCTCTACTGAACCAGGTACTTCTACTGAACCATCTGAGCCGGGCAG
  		CGCAGGTAGCGAACCAGCTACTTCTGGCACTGAACCATCAGGTACTTCTACTGA
  		ACCATCCGAACCAGGTAGCGCAGGTAGCGAACCTGCTACCTCTGGTACTGAGCC
  		ATCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGA
  		ACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAG
  		CGCAGGTACTTCTACTGAACCATCCGAGCCGGGTAGCGCAGGTACTTCCACTGA
  		ACCATCTGAACCTGGTAGCGCAGGTACTTCCACTGAACCATCCGAACCAGGTAG
  		CGCAGGTACTAGCGAACCATCCACCTCCGAACCAGGCGCAGGTAGCGGTGCATC
  		TGAACCGACTTCTACTGAACCAGGTACTTCCACTGAACCATCTGAGCCAGGTAG
  		CGCAGGTACTTCCACCGAACCATCCGAACCAGGTAGCGCAGGTACTTCCACCGA
  		ACCATCCGAACCTGGCAGCGCAGGTAGCGAACCGGCAACCTCTGGTACTGAACC
  		ATCAGGTAGCGGTGCATCCGAGCCGACCTCTACTGAACCAGGTAGCGAACCAGC
  		AACTTCTGGCACTGAGCCATCAGGTAGCGAACCAGCTACCTCTGGTACTGAACC
  		ATCAGGTAGCGAACCGGCAACCTCTGGCACTGAGCCATCAGGTAGCGAACCAG
  		CAACTTCTGGTACTGAACCATCAGGTACTAGCGAGCCATCTACTTCCGAACCAG
  		GTGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCAT
  		CTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGCA
  		GCGCAGGTAGCGAACCTGCAACCTCCGGCACTGAGCCATCAGGTAGCGGCGCA
  		TCTGAACCAACCTCTACTGAACCAGGTACTTCCACCGAACCATCTGAGCCAGGC
  		AGCGCA
  BD864	127	GGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGCA
  		ACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGCA
  		GGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGCTAC
  		CTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAACTGC
  		AGGTACTTCCACTGAAGCAAGTGAAGGCTCCGCATCAGGTACTTCCACCGAAGC
  		AAGCGAAGGCTCCGCATCAGGTACTAGTGAGTCCGCAACTAGCGAATCCGGTGC
  		AGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGC
  		TAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGC
  		AGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGC
  		TACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGC
  		AGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGC
  		TAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGC
  		AGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGC
  		TAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGC
  		AGGTACTAGCGAGTCCGCTACTAGCGAATCTGGCGCAGGTACTTCCACTGAAGC
  		TAGTGAAGGTTCTGCATCAGGTAGCGAAACTGCTACTTCTGGTTCCGAAACTGC
  		AGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGTTC
  		CGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACTGC
  		AGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGC
  		TACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGC
  		AGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCCGC
  		TACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACTGC
  		AGGTAGCGAAACCGCTACCTCTGGTTCCGAAACTGCAGGTACTTCTACCGAGGC
  		TAGCGAAGGTTCTGCATCAGGTAGCACTGCTGGTTCCGAGACTTCTACTGAAGC
  		AGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTAGTGAATCCGC
  		AACTAGCGAATCTGGCGCAGGTAGCACTGCAGGCTCTGAGACTTCCACTGAAGC
  		AGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTC
  		TGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATC
  		AGGTAGCACTGCTGGTTCCGAAACCTCTACCGAAGCAGGTAGCACTGCAGGTTC
  		TGAAACCTCCACTGAAGCAGGTACTTCCACTGAGGCTAGTGAAGGCTCTGCATC
  		AGGTAGCACTGCAGGTTCTGAGACTTCCACCGAAGCAGGTAGCGAAACTGCTAC
  		TTCTGGTTCCGAAACTGCAGGTACTTCCACTGAAGCTAGTGAAGGTTCCGCATC
  		AGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCAA
  		CCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGCG
  		CAGGTACTAGTGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACCGCA
  		ACCTCCGGTTCTGAAACTGCAGGTACTAGCGAATCCGCAACCAGCGAATCTGGC
  		GCAGGTAGCGAAACTGCTACTTCCGGCTCTGAGACTGCAGGTACTTCCACCGAA
  		GCAAGCGAAGGTTCCGCATCAGGTACTTCCACCGAGGCTAGTGAAGGCTCTGCA
  		TCAGGTAGCACTGCTGGCTCCGAGACTTCTACCGAAGCAGGTAGCACTGCAGGT
  		TCCGAAACTTCCACTGAAGCAGGTAGCGAAACTGCTACCTCTGGCTCTGAGACT
  		GCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATCC
  		GCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGACT
  		GCAGGTAGCGAAACTGCTACTTCCGGCTCCGAGACTGCAGGTAGCGAAACTGCT
  		ACTTCTGGCTCCGAAACTGCAGGTACTTCTACTGAGGCTAGTGAAGGTTCCGCA
  		TCAGGTACTAGCGAGTCCGCAACCAGCGAATCCGGCGCAGGTAGCGAAACTGC
  		TACCTCTGGCTCCGAGACTGCAGGTAGCGAAACTGCAACCTCTGGCTCTGAAAC
  		TGCAGGTACTAGCGAATCTGCTACTAGCGAATCCGGCGCAGGTACTAGCGAATC
  		CGCTACCAGCGAATCCGGCGCAGGTAGCGAAACTGCAACCTCTGGTTCCGAGAC
  		TGCA

• One may clone the library of XTEN-encoding genes into one or more expression vectors known in the art. To facilitate the identification of well-expressing library members, one can construct the library as fusion to a reporter protein. Non-limiting examples of suitable reporter genes are green fluorescent protein, luciferace, alkaline phosphatase, and beta-galactosidase. By screening, one can identify short XTEN sequences that can be expressed in high concentration in the host organism of choice. Subsequently, one can generate a library of random XTEN dimers and repeat the screen for high level of expression. Subsequently, one can screen the resulting constructs for a number of properties such as level of expression, protease stability, or binding to antiserum.
• One aspect of the invention is to provide polynucleotide sequences encoding the components of the fusion protein wherein the creation of the sequence has undergone codon optimization. Of particular interest is codon optimization with the goal of improving expression of the polypeptide compositions and to improve the genetic stability of the encoding gene in the production hosts. For example, codon optimization is of particular importance for XTEN sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization is performed using computer programs (Gustafsson, C., et al. (2004)Trends Biotechnol,22: 346-53), some of which minimize ribosomal pausing (Coda Genomics Inc.). In one embodiment, one can perform codon optimization by constructing codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. When designing XTEN sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (e.g. the AGG and AGA arginine codons and one leucine codon inE. coli). In the case ofE. coli, two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thus codon optimization of the gene encoding XTEN sequences can be very desirable. DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels. Thus, when possible, it is preferred to choose codons such that the GC-content of XTEN-encoding sequence is suitable for the production organism that will be used to manufacture the XTEN.
• Optionally, the full-length XTEN-encoding gene comprises one or more sequencing islands. In this context, sequencing islands are short-stretch sequences that are distinct from the XTEN library construct sequences and that include a restriction site not present or expected to be present in the full-length XTEN-encoding gene. In one embodiment, a sequencing island is thesequence 5′-AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGT-3′ (SEQ ID NO: 128). In another embodiment, a sequencing island is thesequence 5′-AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGT-3′ (SEQ ID NO: 129).
• In one embodiment, polynucleotide libraries are constructed using the disclosed methods wherein all members of the library encode the same amino acid sequence but where codon usage for the respective amino acids in the sequence is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products.
• Optionally, one can sequence clones in the library to eliminate isolates that contain undesirable sequences. The initial library of short XTEN sequences allows some variation in amino acid sequence. For instance one can randomize some codons such that a number of hydrophilic amino acids can occur in a particular position. During the process of iterative multimerization one can screen the resulting library members for other characteristics like solubility or protease resistance in addition to a screen for high-level expression.
• Once the gene that encodes the XTEN of desired length and properties is selected, it is genetically fused at the desired location to the nucleotides encoding the CF gene(s) by cloning it into the construct adjacent and in frame with the gene coding for CF, or alternatively between nucleotides encoding adjacent domains of the CF, or alternatively within a sequence encoding a given CF domain, or alternatively in frame with nucleotides encoding a spacer/cleavage sequence linked to a terminal XTEN. The invention provides various permutations of the foregoing, depending on the CFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion protein comprising a CF and two XTEN, such as embodied by formula VI, as depicted above, the gene would have polynucleotides encoding CF, encoding two XTEN, which can be identical or different in composition and sequence length. In one non-limiting embodiment of the foregoing, the CF polynucleotides would encode coagulation factor and the polynucleotides encoding the C-terminus XTEN would encode AE864 and the polynucleotides encoding an internal XTEN adjacent to the C-terminus of EGF2 would encode AE144. The step of cloning the CF genes into the XTEN construct can occur through a ligation or multimerization step, as shown inFIG. 32. The constructs encoding CFXTEN fusion proteins can be designed in different configurations of the components XTEN, CF, and spacer sequences, such as the configurations of formulae I-VI. In one embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5′ to 3′) CF and XTEN. In another embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5′ to 3′) CF, spacer sequence, and XTEN. The spacer polynucleotides can optionally comprise sequences encoding cleavage sequences. As will be apparent to those of skill in the art, other permutations or multimers of the foregoing are possible.
• The invention also encompasses polynucleotides comprising XTEN-encoding polynucleotide variants that have a high percentage of sequence identity compared to (a) a polynucleotide sequence from Table 8, or (b) sequences that are complementary to the polynucleotides of (a). A polynucleotide with a high percentage of sequence identity is one that has at least about an 80% nucleic acid sequence identity, alternatively at least about 81%, alternatively at least about 82%, alternatively at least about 83%, alternatively at least about 84%, alternatively at least about 85%, alternatively at least about 86%, alternatively at least about 87%, alternatively at least about 88%, alternatively at least about 89%, alternatively at least about 90%, alternatively at least about 91%, alternatively at least about 92%, alternatively at least about 93%, alternatively at least about 94%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, and alternatively at least about 99% nucleic acid sequence identity compared to (a) or (b) of the foregoing, or that can hybridize with the target polynucleotide or its complement under stringent conditions.
• Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may also be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics. 1981. 2: 482-489), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, (Journal of Molecular Biology. 1970. 48:443-453). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores.
• Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the polynucleotides that encode the CFXTEN sequences under stringent conditions, such as those described herein.
• The resulting polynucleotides encoding the CFXTEN chimeric fusion proteins can then be individually cloned into an expression vector. The nucleic acid sequence is inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientific and patent literature.
• Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• The invention provides for the use of plasmid vectors containing replication and control sequences that are compatible with and recognized by the host cell, and are operably linked to the CFXTEN gene for controlled expression of the CFXTEN fusion proteins. The vector ordinarily carries a replication site, as well as sequences that encode proteins that are capable of providing phenotypic selection in transformed cells. Such vector sequences are well known for a variety of bacteria, yeast, and viruses. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. “Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA encoding the fusion protein in a suitable host. The requirements are that the vectors are replicable and viable in the host cell of choice. Low- or high-copy number vectors may be used as desired.
• Other suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col EI, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or the expression control sequences; and the like. Yeast expression systems that can also be used in the present invention include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.
• The control sequences of the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
• Examples of suitable promoters for directing the transcription of the DNA encoding the CF polypeptide variant in mammalian cells are the SV40 promoter (Subramani et al.,Mol. Cell. Biol.1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.,Science222 (1983), 809-814), the CMV promoter (Boshart et al.,Cell41:521-530, 1985) or theadenovirus 2 major late promoter (Kaufman and Sharp,Mol. Cell. Biol,2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
• Examples of suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter. Examples of other useful promoters are those derived from the gene encodingA. oryzaeTAKA amylase,Rhizomucor mieheiaspartic proteinase,A. nigerneutral a-amylase,A. nigeracid stable α-amylase,A. nigerorA. awamoriglucoamylase(gluA),Rhizomucor mieheilipase,A. oryzaealkaline protease,A. oryzaetriose phosphate isomerase orA. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters.
• Promoters suitable for use in expression vectors with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all is operably linked to the DNA encoding CFXTEN polypeptides. Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, operably linked to the DNA encoding CFXTEN polypeptides.
• The invention contemplates use of other expression systems including, for example, a baculovirus expression system with both non-fusion transfer vectors, such as, but not limited to pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen), pVL1392 (Summers, et al., Virology 84:390-402 (1978) and Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer vectors such as, but not limited to, pAc700 (Summers, et al., Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700, with different reading frames), pAc360 Invitrogen) and pBlueBacHisA, B, C (Invitrogen) can be used.
• Examples of suitable promoters for directing the transcription of the DNA encoding the CF polypeptide variant in mammalian cells are the CMV promoter (Boshart et al.,Cell41:521-530, 1985), the SV40 promoter (Subramani et al.,Mol. Cell. Biol.1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.,Science222 (1983), 809-814), theadenovirus 2 major late promoter (Kaufman and Sharp,Mol. Cell. Biol,2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
• Examples of suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter.
• The DNA sequences encoding the CFXTEN may also, if necessary, be operably connected to a suitable terminator, such as the hGH terminator (Palmiter et al.,Science222, 1983, pp. 809-814) or the TPI1 terminators (Alber and Kawasaki,J. Mol. Appl. Gen.1, 1982, pp. 419-434) or ADH3 (McKnight et al.,The EMBO J.4, 1985, pp. 2093-2099). Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the CFXTEN sequence itself, including splice sites obtained from adenovirus. Also contained in the expression vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from theadenovirus 5 E1b region, the hGH terminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981). The expression vectors may also include a noncoding viral leader sequence, such as theadenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
• To direct the CFXTEN of the present invention into the secretory pathway of the host cells, a secretory signal sequence (a.k.a., a leader sequence, a prepro sequence, or a pre sequence) may be included in the recombinant vector. The secretory signal sequence is operably linked to the DNA sequences encoding the CFXTEN, usually positioned 5′ to the DNA sequence encoding the CFXTEN fusion protein. The secretory signal sequence may be that, normally associated with the protein or may be from a gene encoding another secreted protein. Non-limiting examples include OmpA, PhoA, and DsbA forE. coliexpression, ppL-alpha, DEX4, invertase signal peptide, acid phosphatase signal peptide, CPY, or INU1 for yeast expression, and IL2L, SV40, IgG kappa and IgG lambda for mammalian expression. Signal sequences are typically proteolytically removed from the protein during the translocation and secretion process, generating a defined N-terminus. Methods are disclosed in Arnau, et al., Protein Expression and Purification 48: 1-13 (2006).
• The procedures used to ligate the DNA sequences coding for the CFXTEN, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
• In other cases, the invention provides constructs and methods of making constructs comprising an polynucleotide sequence optimized for expression that encodes at least about 20 to about 60 amino acids with XTEN characteristics that can be included at the N-terminus of an XTEN carrier encoding sequence (in other words, the polynucleotides encoding the 20-60 encoded optimized amino acids are linked in frame to polynucleotides encoding an XTEN component that is N-terminal to CF) to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain. In an advantage of the foregoing, the sequence does not require subsequent cleavage, thereby reducing the number of steps to manufacture XTEN-containing compositions. As described in more detail in the Examples, the optimized N-terminal sequence has attributes of an unstructured protein, but may include nucleotide bases encoding amino acids selected for their ability to promote initiation of translation and enhanced expression. In one embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE912. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM923. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AE48. In another embodiment of the foregoing, the optimized polynucleotide encodes an XTEN sequence with at least about 90% sequence identity compared to AM48. In one embodiment, the optimized polynucleotide NTS comprises a sequence that exhibits at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity compared to a sequence or its complement selected from

• (SEQ ID NO: 130)

  AE 48: 5′-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAG

  GTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC

  TTCTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTAC

  CGGTTCTCCA-3′

  and

  (SEQ ID NO: 131)

  AM 48: 5′-ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAG

  GTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCC

  CGTCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCTA

  CTGGCTCTCCA-3′

• In this manner, a chimeric DNA molecule coding for a monomeric CFXTEN fusion protein is generated within the construct. Optionally, this chimeric DNA molecule may be transferred or cloned into another construct that is a more appropriate expression vector. At this point, a host cell capable of expressing the chimeric DNA molecule can be transformed with the chimeric DNA molecule.
• Examples of mammalian cell lines for use in the present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al.,J. Gen. Virol.36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-K1 (ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293-F (Invitrogen R790-7). A tk⁻ts13 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used within the present invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin,Proc. Natl. Acad. Sci. USA77:4216-4220, 1980).
• Examples of suitable yeasts cells include cells ofSaccharomycesspp. orSchizosaccharomycesspp., in particular strains ofSaccharomyces cerevisiaeorSaccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos. 4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences encoding the CFXTEN may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above. Further examples of suitable yeast cells are strains ofKluyveromyces, such asK. lactis, Hansenula, e.g.H. polymorpha, orPichia, e.g.P. pastoris(cf. Gleeson et al.,J. Gen. Microbiol.132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279). Examples of other fungal cells are cells of filamentous fungi, e.g.Aspergillusspp.,Neurosporaspp.,Fusariumspp. orTrichodermaspp., in particular strains ofA. oryzae, A. nidulansorA. niger. The use ofAspergillusspp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438 The transformation ofF. oxysporummay, for instance, be carried out as described by Malardier et al., 1989,Gene78: 147-156. The transformation ofTrichodermaspp. may be performed for instance as described in EP 244 234.
• Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such asEscherichia coli, (e.g., strain DH5-α),Bacillus subtilis, Salmonella typhimurium, or strains of the genera ofPseudomonas, StreptomycesandStaphylococcus. Non-limiting examples of suitable prokaryotes include those from the genera:Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus; Paracoccus; Pseudomonas; Staphylococcus; Streptococcus; Streptomyces; Thermoplasma; andVibrio.
• Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g., Kaufman and Sharp,J. Mol. Biol.159 (1982), 601-621; Southern and Berg,J. Mol. Appl. Genet.1 (1982), 327-341; Loyter et al.,Proc. Natl. Acad. Sci. USA79 (1982), 422-426; Wigler et al.,Cell14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics7 (1981), 603, Graham and van der Eb,Virology52 (1973), 456; and Neumann et al.,EMBO J.1 (1982), 841-845.
• Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973), transfection with many commercially available reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) or lipofectamine (Invitrogen) or by electroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells that express the exogenous DNA, a gene that confers a selectable phenotype (a selectable marker) is generally introduced into cells along with the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence. Further examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., incorporated herein by reference). The person skilled in the art will easily be able to choose suitable selectable markers. Any known selectable marker may be employed so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.
• Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If, on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as “carrier DNA,” to the mixture that is introduced into the cells.
• After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to begin expressing the gene of interest. As used herein the term “appropriate growth medium” means a medium containing nutrients and other components required for the growth of cells and the expression of the CFXTEN of interest. Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors. For production of gamma-carboxylated proteins, the medium will contain vitamin K, preferably at a concentration of about 0.1 μg/ml to about 5 μg/ml. Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the CF polypeptide variant of interest.
• The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the CF polypeptide variant after which the resulting peptide may be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
• Gene expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
• Gene expression, alternatively, may be measured by immunological of fluorescent methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids or the detection of selectable markers, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence CF polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to CF and encoding a specific antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).
• Expressed CFXTEN polypeptide product(s) may be purified via methods known in the art or by methods disclosed herein. Procedures such as gel filtration, affinity purification (e.g., using an anti-CF antibody column), salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography and gel electrophoresis may be used; each tailored to recover and purify the fusion protein produced by the respective host cells. Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography. Some expressed CFXTEN may require refolding during isolation and purification. Methods of purification are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it is preferred that the CFXTEN fusion proteins of the invention are substantially pure. Thus, in a preferred embodiment of the invention the CFXTEN of the invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Purity may be assessed by, e.g., gel electrophoresis, HPLC, and amino-terminal amino acid sequencing.
VIII). Pharmaceutical Compositions
• The present invention provides pharmaceutical compositions comprising CFXTEN. In one embodiment, the pharmaceutical composition comprises the CFXTEN fusion protein and at least one pharmaceutically acceptable carrier. CFXTEN polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is combined in admixture with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions or buffers, pharmaceutically acceptable suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions.
• The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
• More particularly, the present pharmaceutical compositions may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, subcutaneous by infusion pump, intramuscular, intravenous and intradermal), intravitreal, and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
• In one embodiment, the pharmaceutical composition is administered subcutaneously. In this embodiment, the composition may be supplied as a lyophilized powder to be reconstituted prior to administration. The composition may also be supplied in a liquid form, which can be administered directly to a patient. In one embodiment, the composition is supplied as a liquid in a pre-filled syringe such that a patient can easily self-administer the composition.
• Extended release formulations useful in the present invention may be oral formulations comprising a matrix and a coating composition. Suitable matrix materials may include waxes (e.g., carnauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols), oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow, palm oil, and soya bean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and polyethylene glycol). Other suitable matrix tabletting materials are microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, ethyl cellulose, with other carriers, and fillers. Tablets may also contain granulates, coated powders, or pellets. Tablets may also be multi-layered. Multi-layered tablets are especially preferred when the active ingredients have markedly different pharmacokinetic profiles. Optionally, the finished tablet may be coated or uncoated.
• The coating composition may comprise an insoluble matrix polymer and/or a water soluble material. Water soluble materials can be polymers such as polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, or monomeric materials such as sugars (e.g., lactose, sucrose, fructose, mannitol and the like), salts (e.g., sodium chloride, potassium chloride and the like), organic acids (e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), and mixtures thereof. Optionally, an enteric polymer may be incorporated into the coating composition. Suitable enteric polymers include hydroxypropyl methyl cellulose, acetate succinate, hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, shellac, zein, and polymethacrylates containing carboxyl groups. The coating composition may be plasticised by adding suitable plasticisers such as, for example, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, acetylated citrate esters, dibutylsebacate, and castor oil. The coating composition may also include a filler, which can be an insoluble material such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or polacrilin potassium. The coating composition may be applied as a solution or latex in organic solvents or aqueous solvents or mixtures thereof. Solvents such as water, lower alcohol, lower chlorinated hydrocarbons, ketones, or mixtures thereof may be used.
• The compositions of the invention may be formulated using a variety of excipients. Suitable excipients include microcrystalline cellulose (e.g. Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR Methocel K100M, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®), and protamine sulfate. The slow release agent may also comprise a carrier, which can comprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Pharmaceutically acceptable salts can also be used in these slow release agents, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes may also be used as a carrier.
• In another embodiment, the compositions of the present invention are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time. Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. In one embodiment, the liposome may be coated with a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the contents of which are incorporated by reference in their entirety.
• Liposomes may be comprised of any lipid or lipid combination known in the art. For example, the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1 [(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3 [N—(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); or dimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the proper range to impart stability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and 5,874,104.
• Additional liposomal technologies are described in U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479, the contents of which are incorporated herein by reference. These describe liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a liposome for the extended release of the polypeptides of the present invention.
• For liquid formulations, a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.
• Administration via transdermal formulations can be performed using methods also known in the art, including those described generally in, e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770, 4,861,800, 6,743,211, 6,945,952, 4,284,444, and WO 89/09051, incorporated herein by reference in their entireties. A transdermal patch is a particularly useful embodiment with polypeptides having absorption problems. Patches can be made to control the release of skin-permeable active ingredients over a 12 hour, 24 hour, 3 day, and 7 day period. In one example, a 2-fold daily excess of a polypeptide of the present invention is placed in a non-volatile fluid. The compositions of the invention are provided in the form of a viscous, non-volatile liquid. The penetration through skin of specific formulations may be measures by standard methods in the art (for example, Franz et al., J. Invest. Derm. 64:194-195 (1975)). Examples of suitable patches are passive transfer skin patches, iontophoretic skin patches, or patches with microneedles such as Nicoderm.
• In other embodiments, the composition may be delivered via intranasal, buccal, or sublingual routes to the brain to enable transfer of the active agents through the olfactory passages into the CNS and reducing the systemic administration. Devices commonly used for this route of administration are included in U.S. Pat. No. 6,715,485. Compositions delivered via this route may enable increased CNS dosing or reduced total body burden reducing systemic toxicity risks associated with certain drugs. Preparation of a pharmaceutical composition for delivery in a subdermally implantable device can be performed using methods known in the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518; 5,660,848; and 5,756,115.
• Osmotic pumps may be used as slow release agents in the form of tablets, pills, capsules or implantable devices. Osmotic pumps are well known in the art and readily available to one of ordinary skill in the art from companies experienced in providing osmotic pumps for extended release drug delivery. Examples are ALZA's DUROS™; ALZA's OROS™; Osmotica Pharmaceutical's Osmodex™ system; Shire Laboratories' EnSoTrol™ system; and Alzet™. Patents that describe osmotic pump technology are U.S. Pat. Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532; 6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776; 4,200,0984; and 4,088,864, the contents of which are incorporated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce an osmotic pump for the extended release of the polypeptides of the present invention.
• Syringe pumps may also be used as slow release agents. Such devices are described in U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the contents of which are incorporated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a syringe pump for the extended release of the compositions of the present invention.
IX). Pharmaceutical Kits
• In another aspect, the invention provides a kit to facilitate the use of the CFXTEN polypeptides. The kit comprises the pharmaceutical composition provided herein, a label identifying the pharmaceutical composition, and an instruction for storage, reconstitution and/or administration of the pharmaceutical compositions to a subject. In some embodiment, the kit comprises, preferably: (a) an amount of a CFXTEN fusion protein composition sufficient to treat a disease, condition or disorder upon administration to a subject in need thereof; and (b) an amount of a pharmaceutically acceptable carrier; together in a formulation ready for injection or for reconstitution with sterile water, buffer, or dextrose; together with a label identifying the CFXTEN drug and storage and handling conditions, and a sheet of the approved indications for the drug, instructions for the reconstitution and/or administration of the CFXTEN drug for the use for the prevention and/or treatment of a approved indication, appropriate dosage and safety information, and information identifying the lot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a second container that can carry a suitable diluent for the CFXTEN composition, the use of which will provide the user with the appropriate concentration of CFXTEN to be delivered to the subject.
EXAMPLESExample 1Construction of XTEN_AD36 Motif Segments
• The following example describes the construction of a collection of codon-optimized genes encoding motif sequences of 36 amino acids. As a first step, a stuffer vector pCW0359 was constructed based on a pET vector and that includes a T7 promoter. pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease recognition site followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion. The stuffer sequence is followed by a truncated version of the GFP gene and a His tag. The stuffer sequence contains stop codons and thusE. colicells carrying the stuffer plasmid pCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification. The sequences were designated XTEN_AD36, reflecting the AD family of motifs. Its segments have the amino acid sequence [X]₃where X is a 12mer peptide with the sequences: GESPGGSSGSES (SEQ ID NO: 31), GSEGSSGPGESS (SEQ ID NO: 32), GSSESGSSEGGP (SEQ ID NO: 33), or GSGGEPSESGSS (SEQ ID NO: 34). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

• (SEQ ID NO: 132)

  	AD1for: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC

  (SEQ ID NO: 133)

  	AD1rev: ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC

  (SEQ ID NO: 134)

  	AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC

  (SEQ ID NO: 135)

  	AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT

  (SEQ ID NO: 136)

  	AD3for: AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC

  (SEQ ID NO: 137)

  	AD3rev: ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA

  (SEQ ID NO: 138)

  AD4for: AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC

• We also annealed the phosphorylatedoligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and thenon-phosphorylated oligonucleotide pr_—3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stiffer vector pCW0359. Most of the clones in the resulting library designated LCW0401 showed green fluorescence after induction, which shows that the sequence of XTEN_AD36 had been ligated in frame with the GFP gene and that most sequences of XTEN_AD36 had good expression levels.
• We screened 96 isolates from library LCW0401 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 39 clones were identified that contained correct XTEN_AD36 segments. The file names of the nucleotide and amino acid constructs for these segments are listed in Table 9.

• TABLE 9
  DNA and Amino Acid Sequences for 36-mer motifs

  		SEQ		SEQ
  		ID		ID
  File name	Ammo acid sequence	NO:	Nucleotide sequence	NO:
  LCW0401_001_GFP-	GSGGEPSESGSSGESPGG	141	GGTTCTGGTGGCGAACCGTCCGAGTC	142
  N_A01.ab1	SSGSESGESPGGSSGSES		TGGTAGCTCAGGTGAATCTCCGGGTG
  			GCTCTAGCGGTTCCGAGTCAGGTGAA
  			TCTCCTGGTGGTTCCAGCGGTTCCGA
  			GTCA
  LCW0401_002_GFP-	GSEGSSGPGESSGESPGG	143	GGTAGCGAAGGTTCTTCTGGTCCTGG	144
  N_B01.ab1	SSGSESGSSESGSSEGGP		CGAGTCTTCAGGTGAATCTCCTGGTG
  			GTTCCAGCGGTTCTGAATCAGGTTCC
  			TCCGAAAGCGGTTCTTCCGAGGGCG
  			GTCCA
  LCW0401_003_GFP-	GSSESGSSEGGPGSSESG	145	GGTTCCTCTGAAAGCGGTTCTTCCGA	146
  N_C01.ab1	SSEGGPGESPGGSSGSES		AGGTGGTCCAGGTTCCTCTGAAAGCG
  			GTTCTTCTGAGGGTGGTCCAGGTGAA
  			TCTCCGGGTGGCTCCAGCGGTTCCGA
  			GTCA
  LCW0401_004_GFP-	GSGGEPSESGSSGSSESG	147	GGTTCCGGTGGCGAACCGTCTGAATC	148
  N_D01.ab1	SSEGGPGSGGEPSESGSS		TGGTAGCTCAGGTTCTTCTGAAAGCG
  			GTTCTTCCGAGGGTGGTCCAGGTTCT
  			GGTGGTGAACCTTCCGAGTCTGGTAG
  			CTCA
  LCW0401_007_GFP-	GSSESGSSEGGPGSEGSS	149	GGTTCTTCCGAAAGCGGTTCTTCTGA	150
  N_F01.ab1	GPGESSGSEGSSGPGESS		GGGTGGTCCAGGTAGCGAAGGTTCTT
  			CCGGTCCAGGTGAGTCTTCAGGTAGC
  			GAAGGTTCTTCTGGTCCTGGTGAATC
  			TTCA
  LCW0401_008_GFP-	GSSESGSSEGGPGESPGG	151	GGTTCCTCTGAAAGCGGTTCTTCCGA	152
  N_G01.ab1	SSGSESGSEGSSGPGESS		GGGTGGTCCAGGTGAATCTCCAGGT
  			GGTTCCAGCGGTTCTGAGTCAGGTAG
  			CGAAGGTTCTTCTGGTCCAGGTGAAT
  			CCTCA
  LCW0401_012_GFP-	GSGGEPSESGSSGSGGEP	153	GGTTCTGGTGGTGAACCGTCTGAGTC	154
  N_H01.ab1	SESGSSGSEGSSGPGESS		TGGTAGCTCAGGTTCCGGTGGCGAAC
  			CATCCGAATCTGGTAGCTCAGGTAGC
  			GAAGGTTCTTCCGGTCCAGGTGAGTC
  			TTCA
  LCW0401_015_GFP-	GSSESGSSEGGPGSEGSS	155	GGTTCTTCCGAAAGCGGTTCTTCCGA	156
  N_A02.ab1	GPGESSGESPGGSSGSES		AGGCGGTCCAGGTAGCGAAGGTTCT
  			TCTGGTCCAGGCGAATCTTCAGGTGA
  			ATCTCCTGGTGGCTCCAGCGGTTCTG
  			AGTCA
  LCW0401_016_GFP-	GSSESGSSEGGPGSSESG	157	GGTTCCTCCGAAAGCGGTTCTTCTGA	158
  N_B02.ab1	SSEGGPGSSESGSSEGGP		GGGCGGTCCAGGTTCCTCCGAAAGC
  			GGTTCTTCCGAGGGCGGTCCAGGTTC
  			TTCTGAAAGCGGTTCTTCCGAGGGCG
  			GTCCA
  LCW0401_020_GFP-	GSGGEPSESGSSGSEGSS	159	GGTTCCGGTGGCGAACCGTCCGAATC	160
  N_E02.ab1	GPGESSGSSESGSSEGGP		TGGTAGCTCAGGTAGCGAAGGTTCTT
  			CTGGTCCAGGCGAATCTTCAGGTTCC
  			TCTGAAAGCGGTTCTTCTGAGGGCGG
  			TCCA
  LCW0401_022_GFP-	GSGGEPSESGSSGSSESG	161	GGTTCTGGTGGTGAACCGTCCGAATC	162
  N_F02.ab1	SSEGGPGSGGEPSESGSS		TGGTAGCTCAGGTTCTTCCGAAAGCG
  			GTTCTTCTGAAGGTGGTCCAGGTTCC
  			GGTGGCGAACCTTCTGAATCTGGTAG
  			CTCA
  LCW0401_024_GFP-	GSGGEPSESGSSGSSESG	163	GGTTCTGGTGGCGAACCGTCCGAATC	164
  N_G02.ab1	SSEGGPGESPGGSSGSES		TGGTAGCTCAGGTTCCTCCGAAAGCG
  			GTTCTTCTGAAGGTGGTCCAGGTGAA
  			TCTCCAGGTGGTTCTAGCGGTTCTGA
  			ATCA
  LCW0401_026_GFP-	GSGGEPSESGSSGESPGG	165	GGTTCTGGTGGCGAACCGTCTGAGTC	166
  N_H02.ab1	SSGSESGSEGSSGPGESS		TGGTAGCTCAGGTGAATCTCCTGGTG
  			GCTCCAGCGGTTCTGAATCAGGTAGC
  			GAAGGTTCTTCTGGTCCTGGTGAATC
  			TTCA
  LCW0401_027_GFP-	GSGGEPSESGSSGESPGG	167	GGTTCCGGTGGCGAACCTTCCGAATC	168
  N_A03.ab1	SSGSESGSGGEPSESGSS		TGGTAGCTCAGGTGAATCTCCGGGTG
  			GTTCTAGCGGTTCTGAGTCAGGTTCT
  			GGTGGTGAACCTTCCGAGTCTGGTAG
  			CTCA
  LCW0401_028_GFP-	GSSESGSSEGGPGSSESG	169	GGTTCCTCTGAAAGCGGTTCTTCTGA	170
  N_B03.ab1	SSEGGPGSSESGSSEGGP		GGGCGGTCCAGGTTCTTCCGAAAGC
  			GGTTCTTCCGAGGGCGGTCCAGGTTC
  			TTCCGAAAGCGGTTCTTCTGAAGGCG
  			GTCCA
  LCW0401_030_GFP-	GESPGGSSGSESGSEGSS	171	GGTGAATCTCCGGGTGGCTCCAGCG	172
  N_C03.ab1	GPGESSGSEGSSGPGESS		GTTCTGAGTCAGGTAGCGAAGGTTCT
  			TCCGGTCCGGGTGAGTCCTCAGGTAG
  			CGAAGGTTCTTCCGGTCCTGGTGAGT
  			CTTCA
  LCW0401_031_GFP-	GSGGEPSESGSSGSGGEP	173	GGTTCTGGTGGCGAACCTTCCGAATC	174
  N_D03.ab1	SESGSSGSSESGSSEGGP		TGGTAGCTCAGGTTCCGGTGGTGAAC
  			CTTCTGAATCTGGTAGCTCAGGTTCT
  			TCTGAAAGCGGTTCTTCCGAGGGCGG
  			TCCA
  LCW0401_033_GFP-	GSGGEPSESGSSGSGGEP	175	GGTTCCGGTGGTGAACCTTCTGAATC	176
  N_E03.ab1	SESGSSGSGGEPSESGSS		TGGTAGCTCAGGTTCCGGTGGCGAAC
  			CATCCGAGTCTGGTAGCTCAGGTTCC
  			GGTGGTGAACCATCCGAGTCTGGTA
  			GCTCA
  LCW0401_037_GFP-	GSGGEPSESGSSGSSESG	177	GGTTCCGGTGGCGAACCTTCTGAATC	178
  N_F03.ab1	SSEGGPGSEGSSGPGESS		TGGTAGCTCAGGTTCCTCCGAAAGCG
  			GTTCTTCTGAGGGCGGTCCAGGTAGC
  			GAAGGTTCTTCTGGTCCGGGCGAGTC
  			TTCA
  LCW0401_038_GFP-	GSGGEPSESGSSGSEGSS	179	GGTTCCGGTGGTGAACCGTCCGAGTC	180
  N_G03.ab1	GPGESSGSGGEPSESGSS		TGGTAGCTCAGGTAGCGAAGGTTCTT
  			CTGGTCCGGGTGAGTCTTCAGGTTCT
  			GGTGGCGAACCGTCCGAATCTGGTA
  			GCTCA
  LCW0401_039_GFP-	GSGGEPSESGSSGESPGG	181	GGTTCTGGTGGCGAACCGTCCGAATC	182
  N_H03.ab1	SSGSESGSGGEPSESGSS		TGGTAGCTCAGGTGAATCTCCTGGTG
  			GTTCCAGCGGTTCCGAGTCAGGTTCT
  			GGTGGCGAACCTTCCGAATCTGGTAG
  			CTCA
  LCW0401_040_GFP-	GSSESGSSEGGPGSGGEP	183	GGTTCTTCCGAAAGCGGTTCTTCCGA	184
  N_A04.ab1	SESGSSGSSESGSSEGGP		GGGCGGTCCAGGTTCCGGTGGTGAA
  			CCATCTGAATCTGGTAGCTCAGGTTC
  			TTCTGAAAGCGGTTCTTCTGAAGGTG
  			GTCCA
  LCW0401_042_GFP-	GSEGSSGPGESSGESPGG	185	GGTAGCGAAGGTTCTTCCGGTCCTGG	186
  N_C04.ab1	SSGSESGSEGSSGPGESS		TGAGTCTTCAGGTGAATCTCCAGGTG
  			GCTCTAGCGGTTCCGAGTCAGGTAGC
  			GAAGGTTCTTCTGGTCCTGGCGAGTC
  			CTCA
  LCW0401_046_GFP-	GSSESGSSEGGPGSSESG	187	GGTTCCTCTGAAAGCGGTTCTTCCGA	188
  N_D04.ab1	SSEGGPGSSESGSSEGGP		AGGCGGTCCAGGTTCTTCCGAAAGC
  			GGTTCTTCTGAGGGCGGTCCAGGTTC
  			CTCCGAAAGCGGTTCTTCTGAGGGTG
  			GTCCA
  LCW0401_047_GFP-	GSGGEPSESGSSGESPGG	189	GGTTCTGGTGGCGAACCTTCCGAGTC	190
  N_E04.ab1	SSGSESGESPGGSSGSES		TGGTAGCTCAGGTGAATCTCCGGGTG
  			GTTCTAGCGGTTCCGAGTCAGGTGAA
  			TCTCCGGGTGGTTCCAGCGGTTCTGA
  			GTCA
  LCW0401_051_GFP-	GSGGEPSESGSSGSEGSS	191	GGTTCTGGTGGCGAACCATCTGAGTC	192
  N_F04.ab1	GPGESSGESPGGSSGSES		TGGTAGCTCAGGTAGCGAAGGTTCTT
  			CCGGTCCAGGCGAGTCTTCAGGTGA
  			ATCTCCTGGTGGCTCCAGCGGTTCTG
  			AGTCA
  LCW0401_053_GFP-	GESPGGSSGSESGESPGG	193	GGTGAATCTCCTGGTGGTTCCAGCGG	194
  N_H04.ab1	SSGSESGESPGGSSGSES		TTCCGAGTCAGGTGAATCTCCAGGTG
  			GCTCTAGCGGTTCCGAGTCAGGTGAA
  			TCTCCTGGTGGTTCTAGCGGTTCTGA
  			ATCA
  LCW0401_054_GFP-	GSEGSSGPGESSGSEGSS	195	GGTAGCGAAGGTTCTTCCGGTCCAGG	196
  N_A05.ab1	GPGESSGSGGEPSESGSS		TGAATCTTCAGGTAGCGAAGGTTCTT
  			CTGGTCCTGGTGAATCCTCAGGTTCC
  			GGTGGCGAACCATCTGAATCTGGTA
  			GCTCA
  LCW0401_059_GFP-	GSGGEPSESGSSGSEGSS	197	GGTTCTGGTGGCGAACCATCCGAATC	198
  N_D05.ab1	GPGESSGESPGGSSGSES		TGGTAGCTCAGGTAGCGAAGGTTCTT
  			CTGGTCCTGGCGAATCTTCAGGTGAA
  			TCTCCAGGTGGCTCTAGCGGTTCCGA
  			ATCA
  LCW0401_060_GFP-	GSGGEPSESGSSGSSESG	199	GGTTCCGGTGGTGAACCGTCCGAATC	200
  N_E05.ab1	SSEGGPGSGGEPSESGSS		TGGTAGCTCAGGTTCCTCTGAAAGCG
  			GTTCTTCCGAGGGTGGTCCAGGTTCC
  			GGTGGTGAACCTTCTGAGTCTGGTAG
  			CTCA
  LCW0401_061_GFP-	GSSESGSSEGGPGSGGEP	201	GGTTCCTCTGAAAGCGGTTCTTCTGA	202
  N_F05.ab1	SESGSSGSEGSSGPGESS		GGGCGGTCCAGGTTCTGGTGGCGAA
  			CCATCTGAATCTGGTAGCTCAGGTAG
  			CGAAGGTTCTTCCGGTCCGGGTGAAT
  			CTTCA
  LCW0401_063_GFP-	GSGGEPSESGSSGSEGSS	203	GGTTCTGGTGGTGAACCGTCCGAATC	204
  N_H05.ab1	GPGESSGSEGSSGPGESS		TGGTAGCTCAGGTAGCGAAGGTTCTT
  			CTGGTCCTGGCGAGTCTTCAGGTAGC
  			GAAGGTTCTTCTGGTCCTGGTGAATC
  			TTCA
  LCW0401_066_GFP-	GSGGEPSESGSSGSSESG	205	GGTTCTGGTGGCGAACCATCCGAGTC	206
  N_B06.ab1	SSEGGPGSGGEPSESGSS		TGGTAGCTCAGGTTCTTCCGAAAGCG
  			GTTCTTCCGAAGGCGGTCCAGGTTCT
  			GGTGGTGAACCGTCCGAATCTGGTA
  			GCTCA
  LCW0401_067_GFP-	GSGGEPSESGSSGESPGG	207	GGTTCCGGTGGCGAACCTTCCGAATC	208
  N_C06.ab1	SSGSESGESPGGSSGSES		TGGTAGCTCAGGTGAATCTCCGGGTG
  			GTTCTAGCGGTTCCGAATCAGGTGAA
  			TCTCCAGGTGGTTCTAGCGGTTCCGA
  			ATCA
  LCW0401_069_GFP-	GSGGEPSESGSSGSGGEP	209	GGTTCCGGTGGTGAACCATCTGAGTC	210
  N_D06.ab1	SESGSSGESPGGSSGSES		TGGTAGCTCAGGTTCCGGTGGCGAAC
  			CGTCCGAGTCTGGTAGCTCAGGTGAA
  			TCTCCGGGTGGTTCCAGCGGTTCCGA
  			ATCA
  LCW0401_070_GFP-	GSEGSSGPGESSGSSESG	211	GGTAGCGAAGGTTCTTCTGGTCCGGG	212
  N_E06.ab1	SSEGGPGSEGSSGPGESS		CGAATCCTCAGGTTCCTCCGAAAGCG
  			GTTCTTCCGAAGGTGGTCCAGGTAGC
  			GAAGGTTCTTCCGGTCCTGGTGAATC
  			TTCA
  LCW0401_078_GFP-	GSSESGSSEGGPGESPGG	213	GGTTCCTCTGAAAGCGGTTCTTCTGA	214
  N_F06.ab1	SSGSESGESPGGSSGSES		AGGCGGTCCAGGTGAATCTCCGGGT
  			GGCTCCAGCGGTTCTGAATCAGGTGA
  			ATCTCCTGGTGGCTCCAGCGGTTCCG
  			AGTCA
  LCW0401_079_GFP-	GSEGSSGPGESSGSEGSS	215	GGTAGCGAAGGTTCTTCTGGTCCAGG	216
  N_G06.ab1	GPGESSGSGGEPSESGSS		CGAGTCTTCAGGTAGCGAAGGTTCTT
  			CCGGTCCTGGCGAGTCTTCAGGTTCC
  			GGTGGCGAACCGTCCGAATCTGGTA
  			GCTCA

Example 2Construction of XTEN_AE36 Segments
• • A codon library encoding XTEN sequences of 36 amino acid length was constructed. The XTEN sequence was designated XTEN_AE36. Its segments have the amino acid sequence [X]₃where X is a 12mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 35), GSEPATSGSE TP (SEQ ID NO: 36), GTSESA TPESGP (SEQ ID NO: 37), or GTSTEPSEGSAP (SEQ ID NO: 38). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

• (SEQ ID NO: 217)

  	AE1for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA

  (SEQ ID NO: 218)

  	AE1rev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT

  (SEQ ID NO: 219)

  	AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC

  (SEQ ID NO: 220)

  	AE2rev: ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT

  (SEQ ID NO: 221)

  	AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC

  (SEQ ID NO: 222)

  	AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT

  (SEQ ID NO: 223)

  	AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC

  (SEQ ID NO: 224)

  AE4rev: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT

• We also annealed the phosphorylatedoligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and thenon-phosphorylated oligonucleotide pr_—3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stiffer vector pCW0359. Most of the clones in the resulting library designated LCW0402 showed green fluorescence after induction which shows that the sequence of XTEN_AE36 had been ligated in frame with the GFP gene and most sequences of XTEN_AE36 show good expression.
• We screened 96 isolates from library LCW0402 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 37 clones were identified that contained correct XTEN_AE36 segments. The file names of the nucleotide and amino acid constructs for these segments are listed in Table 10.

• TABLE 10
  DNA and Amino Acid Sequences for 36-mer motifs

  		SEQ		SEQ
  		ID		ID
  File name	Amino acid sequence	NO:	Nucleotide sequence	NO:
  LCW0402_002_GFP-	GSPAGSPTSTEEGTSE	225	GGTAGCCCGGCAGGCTCTCCGACCTCTA	226
  N_A07.ab1	SATPESGPGTSTEPSE		CTGAGGAAGGTACTTCTGAAAGCGCAAC
  	GSAP		CCCGGAGTCCGGCCCAGGTACCTCTACC
  			GAACCGTCTGAGGGCAGCGCACCA
  LCW0402_003_GFP-	GTSTEPSEGSAPGTST	227	GGTACTTCTACCGAACCGTCCGAAGGCA	228
  N_B07.ab1	EPSEGSAPGTSTEPSE		GCGCTCCAGGTACCTCTACTGAACCTTCC
  	GSAP		GAGGGCAGCGCTCCAGGTACCTCTACCG
  			AACCTTCTGAAGGTAGCGCACCA
  LCW0402_004_GFP-	GTSTEPSEGSAPGTSE	229	GGTACCTCTACCGAACCGTCTGAAGGTA	230
  N_C07.ab1	SATPESGPGTSESATP		GCGCACCAGGTACCTCTGAAAGCGCAAC
  	ESGP		TCCTGAGTCCGGTCCAGGTACTTCTGAAA
  			GCGCAACCCCGGAGTCTGGCCCA
  LCW0402_005_GFP-	GTSTEPSEGSAPGTSE	231	GGTACTTCTACTGAACCGTCTGAAGGTA	231
  N_D07.ab1	SATPESGPGTSESATP		GCGCACCAGGTACTTCTGAAAGCGCAAC
  	ESGP		CCCGGAATCCGGCCCAGGTACCTCTGAA
  			AGCGCAACCCCGGAGTCCGGCCCA
  LCW0402_006_GFP-	GSEPATSGSETPGTSE	233	GGTAGCGAACCGGCAACCTCCGGCTCTG	234
  N_E07.ab1	SATPESGPGSPAGSPT		AAACCCCAGGTACCTCTGAAAGCGCTAC
  	STEE		TCCTGAATCCGGCCCAGGTAGCCCGGCA
  			GGTTCTCCGACTTCCACTGAGGAA
  LCW0402_008_GFP-	GTSESATPESGPGSEP	235	GGTACTTCTGAAAGCGCAACCCCTGAAT	236
  N_F07.ab1	ATSGSETPGTSTEPSE		CCGGTCCAGGTAGCGAACCGGCTACTTC
  	GSAP		TGGCTCTGAGACTCCAGGTACTTCTACCG
  			AACCGTCCGAAGGTAGCGCACCA
  LCW0402_009_GFP-	GSPAGSPTSTEEGSPA	237	GGTAGCCCGGCTGGCTCTCCAACCTCCA	238
  N_G07.ab1	GSPTSTEEGSEPATSG		CTGAGGAAGGTAGCCCGGCTGGCTCTCC
  	SETP		AACCTCCACTGAAGAAGGTAGCGAACCG
  			GCTACCTCCGGCTCTGAAACTCCA
  LCW0402_011_GFP-	GSPAGSPTSTEEGTSE	239	GGTAGCCCGGCTGGCTCTCCTACCTCTAC	240
  N_A08.ab1	SATPESGPGTSTEPSE		TGAGGAAGGTACTTCTGAAAGCGCTACT
  	GSAP		CCTGAGTCTGGTCCAGGTACCTCTACTGA
  			ACCGTCCGAAGGTAGCGCTCCA
  LCW0402_012_GFP-	GSPAGSPTSTEEGSPA	241	GGTAGCCCTGCTGGCTCTCCGACTTCTAC	242
  N_B08.ab1	GSPTSTEEGTSTEPSE		TGAGGAAGGTAGCCCGGCTGGTTCTCCG
  	GSAP		ACTTCTACTGAGGAAGGTACTTCTACCG
  			AACCTTCCGAAGGTAGCGCTCCA
  LCW0402_013_GFP-	GTSESATPESGPGTST	243	GGTACTTCTGAAAGCGCTACTCCGGAGT	244
  N_C08.ab1	EPSEGSAPGTSTEPSE		CCGGTCCAGGTACCTCTACCGAACCGTC
  	GSAP		CGAAGGCAGCGCTCCAGGTACTTCTACT
  			GAACCTTCTGAGGGTAGCGCTCCA
  LCW0402_014_GFP-	GTSTEPSEGSAPGSPA	245	GGTACCTCTACCGAACCTTCCGAAGGTA	246
  N_D08.ab1	GSPTSTEEGTSTEPSE		GCGCTCCAGGTAGCCCGGCAGGTTCTCC
  	GSAP		TACTTCCACTGAGGAAGGTACTTCTACCG
  			AACCTTCTGAGGGTAGCGCACCA
  LCW0402_015_GFP-	GSEPATSGSETPGSPA	247	GGTAGCGAACCGGCTACTTCCGGCTCTG	248
  N_E08.ab1	GSPTSTEEGTSESATP		AGACTCCAGGTAGCCCTGCTGGCTCTCC
  	ESGP		GACCTCTACCGAAGAAGGTACCTCTGAA
  			AGCGCTACCCCTGAGTCTGGCCCA
  LCW0402_016_GFP-	GTSTEPSEGSAPGTSE	249	GGTACTTCTACCGAACCTTCCGAGGGCA	250
  N_F08.ab1	SATPESGPGTSESATP		GCGCACCAGGTACTTCTGAAAGCGCTAC
  	ESGP		CCCTGAGTCCGGCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAATCCGGTCCA
  LCW0402_020_GFP-	GTSTEPSEGSAPGSEP	251	GGTACTTCTACTGAACCGTCTGAAGGCA	252
  N_G08.ab1	ATSGSETPGSPAGSPT		GCGCACCAGGTAGCGAACCGGCTACTTC
  	STEE		CGGTTCTGAAACCCCAGGTAGCCCAGCA
  			GGTTCTCCAACTTCTACTGAAGAA
  LCW0402_023_GFP-	GSPAGSPTSTEEGTSE	253	GGTAGCCCTGCTGGCTCTCCAACCTCCAC	254
  N_A09.ab1	SATPESGPGSEPATSG		CGAAGAAGGTACCTCTGAAAGCGCAACC
  	SETP		CCTGAATCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCCA
  LCW0402_024_GFP-	GTSESATPESGPGSPA	255	GGTACTTCTGAAAGCGCTACTCCTGAGTC	256
  N_B09.ab1	GSPTSTEEGSPAGSPT		CGGCCCAGGTAGCCCGGCTGGCTCTCCG
  	STEE		ACTTCCACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGAA
  LCW0402_025_GFP-	GTSTEPSEGSAPGTSE	257	GGTACCTCTACTGAACCTTCTGAGGGCA	258
  N_C09.ab1	SATPESGPGTSTEPSE		GCGCTCCAGGTACTTCTGAAAGCGCTAC
  	GSAP		CCCGGAGTCCGGTCCAGGTACTTCTACTG
  			AACCGTCCGAAGGTAGCGCACCA
  LCW0402_026_GFP-	GSPAGSPTSTEEGTST	259	GGTAGCCCGGCAGGCTCTCCGACTTCCA	260
  N_D09.ab1	EPSEGSAPGSEPATSG		CCGAGGAAGGTACCTCTACTGAACCTTC
  	SETP		TGAGGGTAGCGCTCCAGGTAGCGAACCG
  			GCAACCTCTGGCTCTGAAACCCCA
  LCW0402_027_GFP-	GSPAGSPTSTEEGTST	261	GGTAGCCCAGCAGGCTCTCCGACTTCCA	262
  N_E09.ab1	EPSEGSAPGTSTEPSE		CTGAGGAAGGTACTTCTACTGAACCTTCC
  	GSAP		GAAGGCAGCGCACCAGGTACCTCTACTG
  			AACCTTCTGAGGGCAGCGCTCCA
  LCW0402_032_GFP-	GSEPATSGSETPGTSE	263	GGTAGCGAACCTGCTACCTCCGGTTCTG	264
  N_H09.ab1	SATPESGPGSPAGSPT		AAACCCCAGGTACCTCTGAAAGCGCAAC
  	STEE		TCCGGAGTCTGGTCCAGGTAGCCCTGCA
  			GGTTCTCCTACCTCCACTGAGGAA
  LCW0402_034_GFP-	GTSESATPESGPGTST	265	GGTACCTCTGAAAGCGCTACTCCGGAGT	266
  N_A10.ab1	EPSEGSAPGTSTEPSE		CTGGCCCAGGTACCTCTACTGAACCGTCT
  	GSAP		GAGGGTAGCGCTCCAGGTACTTCTACTG
  			AACCGTCCGAAGGTAGCGCACCA
  LCW0402_036_GFP-	GSPAGSPTSTEEGTST	267	GGTAGCCCGGCTGGTTCTCCGACTTCCAC	268
  N_C10.ab1	EPSEGSAPGTSTEPSE		CGAGGAAGGTACCTCTACTGAACCTTCT
  	GSAP		GAGGGTAGCGCTCCAGGTACCTCTACTG
  			AACCTTCCGAAGGCAGCGCTCCA
  LCW0402_039_GFP-	GTSTEPSEGSAPGTST	269	GGTACTTCTACCGAACCGTCCGAGGGCA	270
  N_E10.ab1	EPSEGSAPGTSTEPSE		GCGCTCCAGGTACTTCTACTGAACCTTCT
  	GSAP		GAAGGCAGCGCTCCAGGTACTTCTACTG
  			AACCTTCCGAAGGTAGCGCACCA
  LCW0402_040_GFP-	GSEPATSGSETPGTSE	271	GGTAGCGAACCTGCAACCTCTGGCTCTG	272
  N_F10.ab1	SATPESGPGTSTEPSE		AAACCCCAGGTACCTCTGAAAGCGCTAC
  	GSAP		TCCTGAATCTGGCCCAGGTACTTCTACTG
  			AACCGTCCGAGGGCAGCGCACCA
  LCW0402_041_GFP-	GTSTEPSEGSAPGSPA	273	GGTACTTCTACCGAACCGTCCGAGGGTA	274
  N_G10.ab1	GSPTSTEEGTSTEPSE		GCGCACCAGGTAGCCCAGCAGGTTCTCC
  	GSAP		TACCTCCACCGAGGAAGGTACTTCTACC
  			GAACCGTCCGAGGGTAGCGCACCA
  LCW0402_050_GFP-	GSEPATSGSETPGTSE	275	GGTAGCGAACCGGCAACCTCCGGCTCTG	276
  N_A11.ab1	SATPESGPGSEPATSG		AAACTCCAGGTACTTCTGAAAGCGCTAC
  	SETP		TCCGGAATCCGGCCCAGGTAGCGAACCG
  			GCTACTTCCGGCTCTGAAACCCCA
  LCW0402_051_GFP-	GSEPATSGSETPGTSE	277	GGTAGCGAACCGGCAACTTCCGGCTCTG	278
  N_B11.ab1	SATPESGPGSEPATSG		AAACCCCAGGTACTTCTGAAAGCGCTAC
  	SETP		TCCTGAGTCTGGCCCAGGTAGCGAACCT
  			GCTACCTCTGGCTCTGAAACCCCA
  LCW0402_059_GFP-	GSEPATSGSETPGSEP	279	GGTAGCGAACCGGCAACCTCTGGCTCTG	280
  N_E11.ab1	ATSGSETPGTSTEPSE		AAACTCCAGGTAGCGAACCTGCAACCTC
  	GSAP		CGGCTCTGAAACCCCAGGTACTTCTACTG
  			AACCTTCTGAGGGCAGCGCACCA
  LCW0402_060_GFP-	GTSESATPESGPGSEP	281	GGTACTTCTGAAAGCGCTACCCCGGAAT	282
  N_F11.ab1	ATSGSETPGSEPATSG		CTGGCCCAGGTAGCGAACCGGCTACTTC
  	SETP		TGGTTCTGAAACCCCAGGTAGCGAACCG
  			GCTACCTCCGGTTCTGAAACTCCA
  LCW0402_061_GFP-	GTSTEPSEGSAPGTST	283	GGTACCTCTACTGAACCTTCCGAAGGCA	284
  N_G11.ab1	EPSEGSAPGTSESATP		GCGCTCCAGGTACCTCTACCGAACCGTC
  	ESGP		CGAGGGCAGCGCACCAGGTACTTCTGAA
  			AGCGCAACCCCTGAATCCGGTCCA
  LCW0402_065_GFP-	GSEPATSGSETPGTSE	285	GGTAGCGAACCGGCAACCTCTGGCTCTG	286
  N_A12.ab1	SATPESGPGTSESATP		AAACCCCAGGTACCTCTGAAAGCGCTAC
  	ESGP		TCCGGAATCTGGTCCAGGTACTTCTGAA
  			AGCGCTACTCCGGAATCCGGTCCA
  LCW0402_066_GFP-	GSEPATSGSETPGSEP	287	GGTAGCGAACCTGCTACCTCCGGCTCTG	288
  N_B12.ab1	ATSGSETPGTSTEPSE		AAACTCCAGGTAGCGAACCGGCTACTTC
  	GSAP		CGGTTCTGAAACTCCAGGTACCTCTACCG
  			AACCTTCCGAAGGCAGCGCACCA
  LCW0402_067_GFP-	GSEPATSGSETPGTST	289	GGTAGCGAACCTGCTACTTCTGGTTCTGA	290
  N_C12.ab1	EPSEGSAPGSEPATSG		AACTCCAGGTACTTCTACCGAACCGTCC
  	SETP		GAGGGTAGCGCTCCAGGTAGCGAACCTG
  			CTACTTCTGGTTCTGAAACTCCA
  LCW0402_069_GFP-	GTSTEPSEGSAPGTST	291	GGTACCTCTACCGAACCGTCCGAGGGTA	292
  N_D12.ab1	EPSEGSAPGSEPATSG		GCGCACCAGGTACCTCTACTGAACCGTC
  	SETP		TGAGGGTAGCGCTCCAGGTAGCGAACCG
  			GCAACCTCCGGTTCTGAAACTCCA
  LCW0402_073_GFP-	GTSTEPSEGSAPGSEP	293	GGTACTTCTACTGAACCTTCCGAAGGTA	294
  N_F12.ab1	ATSGSETPGSPAGSPT		GCGCTCCAGGTAGCGAACCTGCTACTTCT
  	STEE		GGTTCTGAAACCCCAGGTAGCCCGGCTG
  			GCTCTCCGACCTCCACCGAGGAA
  LCW0402_074_GFP-	GSEPATSGSETPGSPA	295	GGTAGCGAACCGGCTACTTCCGGCTCTG	296
  N_G12.ab1	GSPTSTEEGTSESATP		AGACTCCAGGTAGCCCAGCTGGTTCTCC
  	ESGP		AACCTCTACTGAGGAAGGTACTTCTGAA
  			AGCGCTACCCCTGAATCTGGTCCA
  LCW0402_075_GFP-	GTSESATPESGPGSEP	297	GGTACCTCTGAAAGCGCAACTCCTGAGT	298
  N_H12.ab1	ATSGSETPGTSESATP		CTGGCCCAGGTAGCGAACCTGCTACCTC
  	ESGP		CGGCTCTGAGACTCCAGGTACCTCTGAA
  			AGCGCAACCCCGGAATCTGGTCCA

Example 3Construction of XTEN_AF36 Segments
• A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AF36. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 39), GTSTPESGSASP (SEQ ID NO: 40), GTSPSGESSTAP (SEQ ID NO: 41), or GSTSSTAESPGP (SEQ ID NO: 42). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

• (SEQ ID NO: 299)

  	AF1for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC

  (SEQ ID NO: 300)

  	AF1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA

  (SEQ ID NO: 301)

  	AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC

  (SEQ ID NO: 302)

  	AF2rev: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT

  (SEQ ID NO: 303)

  	AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC

  (SEQ ID NO: 304)

  	AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT

  (SEQ ID NO: 305)

  	AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC

  (SEQ ID NO: 306)

  AF4rev: ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA

• We also annealed the phosphorylatedoligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and thenon-phosphorylated oligonucleotide pr_—3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0403 showed green fluorescence after induction which shows that the sequence of XTEN_AF36 had been ligated in frame with the GFP gene and most sequences of XTEN_AF36 show good expression.
• We screened 96 isolates from library LCW0403 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 44 clones were identified that contained correct XTEN_AF36 segments. The file names of the nucleotide and amino acid constructs for these segments are listed in Table 11.

• TABLE 11
  DNA and Amino Acid Sequences for 36-mer motifs

  		SEQ		SEQ
  		ID		ID
  File name	Amino acid sequence	NO:	Nucleotide sequence	NO:
  LCW0403_004_GFP-	GTSTPESGSASPGTSP	307	GGTACTTCTACTCCGGAAAGCGGTTCCG	308
  N_A01.ab1	SGESSTAPGTSPSGES		CATCTCCAGGTACTTCTCCTAGCGGTGA
  	STAP		ATCTTCTACTGCTCCAGGTACCTCTCCTA
  			GCGGCGAATCTTCTACTGCTCCA
  LCW0403_005_GFP-	GTSPSGESSTAPGSTS	309	GGTACTTCTCCGAGCGGTGAATCTTCTA	310
  N_B01.ab1	STAESPGPGTSPSGES		CCGCACCAGGTTCTACTAGCTCTACCGC
  	STAP		TGAATCTCCGGGCCCAGGTACTTCTCCG
  			AGCGGTGAATCTTCTACTGCTCCA
  LCW0403_006_GFP-	GSTSSTAESPGPGTSP	311	GGTTCCACCAGCTCTACTGCTGAATCTC	312
  N_C01.ab1	SGESSTAPGTSTPESG		CTGGTCCAGGTACCTCTCCTAGCGGTGA
  	SASP		ATCTTCTACTGCTCCAGGTACTTCTACTC
  			CTGAAAGCGGCTCTGCTTCTCCA
  LCW0403_007_GFP-	GSTSSTAESPGPGSTS	313	GGTTCTACCAGCTCTACTGCAGAATCTC	314
  N_D01.ab1	STAESPGPGTSPSGES		CTGGCCCAGGTTCCACCAGCTCTACCGC
  	STAP		AGAATCTCCGGGTCCAGGTACTTCCCCT
  			AGCGGTGAATCTTCTACCGCACCA
  LCW0403_008_GFP-	GSTSSTAESPGPGTSP	315	GGTTCTACTAGCTCTACTGCTGAATCTCC	316
  N_E01.ab1	SGESSTAPGTSTPESG		TGGCCCAGGTACTTCTCCTAGCGGTGAA
  	SASP		TCTTCTACCGCTCCAGGTACCTCTACTCC
  			GGAAAGCGGTTCTGCATCTCCA
  LCW0403_010_GFP-	GSTSSTAESPGPGTST	317	GGTTCTACCAGCTCTACCGCAGAATCTC	318
  N_F01.ab1	PESGSASPGSTSESPS		CTGGTCCAGGTACCTCTACTCCGGAAAG
  	GTAP		CGGCTCTGCATCTCCAGGTTCTACTAGC
  			GAATCTCCTTCTGGCACTGCACCA
  LCW0403_011_GFP-	GSTSSTAESPGPGTST	319	GGTTCTACTAGCTCTACTGCAGAATCTC	320
  N_G01.ab1	PESGSASPGTSTPESG		CTGGCCCAGGTACCTCTACTCCGGAAAG
  	SASP		CGGCTCTGCATCTCCAGGTACTTCTACC
  			CCTGAAAGCGGTTCTGCATCTCCA
  LCW0403_012_GFP-	GSTSESPSGTAPGTSP	321	GGTTCTACCAGCGAATCTCCTTCTGGCA	322
  N_H01.ab1	SGESSTAPGSTSESPS		CCGCTCCAGGTACCTCTCCTAGCGGCGA
  	GTAP		ATCTTCTACCGCTCCAGGTTCTACTAGC
  			GAATCTCCTTCTGGCACTGCACCA
  LCW0403_013_GFP-	GSTSSTAESPGPGSTS	323	GGTTCCACCAGCTCTACTGCAGAATCTC	324
  N_A02.ab1	STAESPGPGTSPSGES		CGGGCCCAGGTTCTACTAGCTCTACTGC
  	STAP		AGAATCTCCGGGTCCAGGTACTTCTCCT
  			AGCGGCGAATCTTCTACCGCTCCA
  LCW0403_014_GFP-	GSTSSTAESPGPGTST	325	GGTTCCACTAGCTCTACTGCAGAATCTC	326
  N_B02.ab1	PESGSASPGSTSESPS		CTGGCCCAGGTACCTCTACCCCTGAAAG
  	GTAP		CGGCTCTGCATCTCCAGGTTCTACCAGC
  			GAATCCCCGTCTGGCACCGCACCA
  LCW0403_015_GFP-	GSTSSTAESPGPGSTS	327	GGTTCTACTAGCTCTACTGCTGAATCTCC	328
  N_C02.ab1	STAESPGPGTSPSGES		GGGTCCAGGTTCTACCAGCTCTACTGCT
  	STAP		GAATCTCCTGGTCCAGGTACCTCCCCGA
  			GCGGTGAATCTTCTACTGCACCA
  LCW0403_017_GFP-	GSTSSTAESPGPGSTS	329	GGTTCTACCAGCTCTACCGCTGAATCTC	330
  N_D02.ab1	ESPSGTAPGSTSSTAE		CTGGCCCAGGTTCTACCAGCGAATCCCC
  	SPGP		GTCTGGCACCGCACCAGGTTCTACTAGC
  			TCTACCGCTGAATCTCCGGGTCCA
  LCW0403_018_GFP-	GSTSSTAESPGPGSTS	331	GGTTCTACCAGCTCTACCGCAGAATCTC	332
  N_E02.ab1	STAESPGPGSTSSTAE		CTGGCCCAGGTTCCACTAGCTCTACCGC
  	SPGP		TGAATCTCCTGGTCCAGGTTCTACTAGC
  			TCTACCGCTGAATCTCCTGGTCCA
  LCW0403_019_GFP-	GSTSESPSGTAPGSTS	333	GGTTCTACTAGCGAATCCCCTTCTGGTA	334
  N_F02.ab1	STAESPGPGSTSSTAE		CTGCTCCAGGTTCCACTAGCTCTACCGC
  	SPGP		TGAATCTCCTGGCCCAGGTTCCACTAGC
  			TCTACTGCAGAATCTCCTGGTCCA
  LCW0403_023_GFP-	GSTSESPSGTAPGSTS	335	GGTTCTACTAGCGAATCTCCTTCTGGTA	336
  N_H02.ab1	ESPSGTAPGSTSESPS		CCGCTCCAGGTTCTACCAGCGAATCCCC
  	GTAP		GTCTGGTACTGCTCCAGGTTCTACCAGC
  			GAATCTCCTTCTGGTACTGCACCA
  LCW0403_024_GFP-	GSTSSTAESPGPGSTS	337	GGTTCCACCAGCTCTACTGCTGAATCTC	338
  N_A03.ab1	STAESPGPGSTSSTAE		CTGGCCCAGGTTCTACCAGCTCTACTGC
  	SPGP		TGAATCTCCGGGCCCAGGTTCCACCAGC
  			TCTACCGCTGAATCTCCGGGTCCA
  LCW0403_025_GFP-	GSTSSTAESPGPGSTS	339	GGTTCCACTAGCTCTACCGCAGAATCTC	340
  N_B03.ab1	STAESPGPGTSPSGES		CTGGTCCAGGTTCTACTAGCTCTACTGCT
  	STAP		GAATCTCCGGGTCCAGGTACCTCCCCTA
  			GCGGCGAATCTTCTACCGCTCCA
  LCW0403_028_GFP-	GSSPSASTGTGPGSST	341	GGTTCTAGCCCTTCTGCTTCCACCGGTAC	342
  N_D03.ab1	PSGATGSPGSSTPSGA		CGGCCCAGGTAGCTCTACTCCGTCTGGT
  	TGSP		GCAACTGGCTCTCCAGGTAGCTCTACTC
  			CGTCTGGTGCAACCGGCTCCCCA
  LCW0403_029_GFP-	GTSPSGESSTAPGTST	343	GGTACTTCCCCTAGCGGTGAATCTTCTA	344
  N_E03.ab1	PESGSASPGSTSSTAE		CTGCTCCAGGTACCTCTACTCCGGAAAG
  	SPGP		CGGCTCCGCATCTCCAGGTTCTACTAGC
  			TCTACTGCTGAATCTCCTGGTCCA
  LCW0403_030_GFP-	GSTSSTAESPGPGSTS	345	GGTTCTACTAGCTCTACCGCTGAATCTC	346
  N_F03.ab1	STAESPGPGTSTPESG		CGGGTCCAGGTTCTACCAGCTCTACTGC
  	SASP		AGAATCTCCTGGCCCAGGTACTTCTACT
  			CCGGAAAGCGGTTCCGCTTCTCCA
  LCW0403_031_GFP-	GTSPSGESSTAPGSTS	347	GGTACTTCTCCTAGCGGTGAATCTTCTA	348
  N_G03.ab1	STAESPGPGTSTPESG		CCGCTCCAGGTTCTACCAGCTCTACTGC
  	SASP		TGAATCTCCTGGCCCAGGTACTTCTACC
  			CCGGAAAGCGGCTCCGCTTCTCCA
  LCW0403_033_GFP-	GSTSESPSGTAPGSTS	349	GGTTCTACTAGCGAATCCCCTTCTGGTA	350
  N_H03.ab1	STAESPGPGSTSSTAE		CTGCACCAGGTTCTACCAGCTCTACTGC
  	SPGP		TGAATCTCCGGGCCCAGGTTCCACCAGC
  			TCTACCGCAGAATCTCCTGGTCCA
  LCW0403_035_GFP-	GSTSSTAESPGPGSTS	351	GGTTCCACCAGCTCTACCGCTGAATCTC	352
  N_A04.ab1	ESPSGTAPGSTSSTAE		CGGGCCCAGGTTCTACCAGCGAATCCCC
  	SPGP		TTCTGGCACTGCACCAGGTTCTACTAGC
  			TCTACCGCAGAATCTCCGGGCCCA
  LCW0403_036_GFP-	GSTSSTAESPGPGTSP	353	GGTTCTACCAGCTCTACTGCTGAATCTC	354
  N_B04.ab1	SGESSTAPGTSTPESG		CGGGTCCAGGTACTTCCCCGAGCGGTGA
  	SASP		ATCTTCTACTGCACCAGGTACTTCTACTC
  			CGGAAAGCGGTTCCGCTTCTCCA
  LCW0403_039_GFP-	GSTSESPSGTAPGSTS	355	GGTTCTACCAGCGAATCTCCTTCTGGCA	356
  N_C04.ab1	ESPSGTAPGTSPSGES		CCGCTCCAGGTTCTACTAGCGAATCCCC
  	STAP		GTCTGGTACCGCACCAGGTACTTCTCCT
  			AGCGGCGAATCTTCTACCGCACCA
  LCW0403_041_GFP-	GSTSESPSGTAPGSTS	357	GGTTCTACCAGCGAATCCCCTTCTGGTA	358
  N_D04.ab1	ESPSGTAPGTSTPESG		CTGCTCCAGGTTCTACCAGCGAATCCCC
  	SASP		TTCTGGCACCGCACCAGGTACTTCTACC
  			CCTGAAAGCGGCTCCGCTTCTCCA
  LCW0403_044_GFP-	GTSTPESGSASPGSTS	359	GGTACCTCTACTCCTGAAAGCGGTTCTG	360
  N_E04.ab1	STAESPGPGSTSSTAE		CATCTCCAGGTTCCACTAGCTCTACCGC
  	SPGP		AGAATCTCCGGGCCCAGGTTCTACTAGC
  			TCTACTGCTGAATCTCCTGGCCCA
  LCW0403_046_GFP-	GSTSESPSGTAPGSTS	361	GGTTCTACCAGCGAATCCCCTTCTGGCA	362
  N_F04.ab1	ESPSGTAPGTSPSGES		CTGCACCAGGTTCTACTAGCGAATCCCC
  	STAP		TTCTGGTACCGCACCAGGTACTTCTCCG
  			AGCGGCGAATCTTCTACTGCTCCA
  LCW0403_047_GFP-	GSTSSTAESPGPGSTS	363	GGTTCTACTAGCTCTACCGCTGAATCTC	364
  N_G04.ab1	STAESPGPGSTSESPS		CTGGCCCAGGTTCCACTAGCTCTACCGC
  	GTAP		AGAATCTCCGGGCCCAGGTTCTACTAGC
  			GAATCCCCTTCTGGTACCGCTCCA
  LCW0403_049_GFP-	GSTSSTAESPGPGSTS	365	GGTTCCACCAGCTCTACTGCAGAATCTC	366
  N_H04.ab1	STAESPGPGTSTPESG		CTGGCCCAGGTTCTACTAGCTCTACCGC
  	SASP		AGAATCTCCTGGTCCAGGTACCTCTACT
  			CCTGAAAGCGGTTCCGCATCTCCA
  LCW0403_051_GFP-	GSTSSTAESPGPGSTS	367	GGTTCTACTAGCTCTACTGCTGAATCTCC	368
  N_A05.ab1	STAESPGPGSTSESPS		GGGCCCAGGTTCTACTAGCTCTACCGCT
  	GTAP		GAATCTCCGGGTCCAGGTTCTACTAGCG
  			AATCTCCTTCTGGTACCGCTCCA
  LCW0403_053_GFP-	GTSPSGESSTAPGSTS	369	GGTACCTCCCCGAGCGGTGAATCTTCTA	370
  N_B05.ab1	ESPSGTAPGSTSSTAE		CTGCACCAGGTTCTACTAGCGAATCCCC
  	SPGP		TTCTGGTACTGCTCCAGGTTCCACCAGC
  			TCTACTGCAGAATCTCCGGGTCCA
  LCW0403_054_GFP-	GSTSESPSGTAPGTSP	371	GGTTCTACTAGCGAATCCCCGTCTGGTA	372
  N_C05.ab1	SGESSTAPGSTSSTAE		CTGCTCCAGGTACTTCCCCTAGCGGTGA
  	SPGP		ATCTTCTACTGCTCCAGGTTCTACCAGCT
  			CTACCGCAGAATCTCCGGGTCCA
  LCW0403_057_GFP-	GSTSSTAESPGPGSTS	373	GGTTCTACCAGCTCTACCGCTGAATCTC	374
  N_D05.ab1	ESPSGTAPGTSPSGES		CTGGCCCAGGTTCTACTAGCGAATCTCC
  	STAP		GTCTGGCACCGCACCAGGTACTTCCCCT
  			AGCGGTGAATCTTCTACTGCACCA
  LCW0403_058_GFP-	GSTSESPSGTAPGSTS	375	GGTTCTACTAGCGAATCTCCTTCTGGCA	376
  N_E05.ab1	ESPSGTAPGTSTPESG		CTGCACCAGGTTCTACCAGCGAATCTCC
  	SASP		GTCTGGCACTGCACCAGGTACCTCTACC
  			CCTGAAAGCGGTTCCGCTTCTCCA
  LCW0403_060_GFP-	GTSTPESGSASPGSTS	377	GGTACCTCTACTCCGGAAAGCGGTTCCG	378
  N_F05.ab1	ESPSGTAPGSTSSTAE		CATCTCCAGGTTCTACCAGCGAATCCCC
  	SPGP		GTCTGGCACCGCACCAGGTTCTACTAGC
  			TCTACTGCTGAATCTCCGGGCCCA
  LCW0403_063_GFP-	GSTSSTAESPGPGTSP	379	GGTTCTACTAGCTCTACTGCAGAATCTC	380
  N_G05.ab1	SGESSTAPGTSPSGES		CGGGCCCAGGTACCTCTCCTAGCGGTGA
  	STAP		ATCTTCTACCGCTCCAGGTACTTCTCCGA
  			GCGGTGAATCTTCTACCGCTCCA
  LCW0403_064_GFP-	GTSPSGESSTAPGTSP	381	GGTACCTCCCCTAGCGGCGAATCTTCTA	382
  N_H05.ab1	SGESSTAPGTSPSGES		CTGCTCCAGGTACCTCTCCTAGCGGCGA
  	STAP		ATCTTCTACCGCTCCAGGTACCTCCCCTA
  			GCGGTGAATCTTCTACCGCACCA
  LCW0403_065_GFP-	GSTSSTAESPGPGTST	383	GGTTCCACTAGCTCTACTGCTGAATCTC	384
  N_A06.ab1	PESGSASPGSTSESPS		CTGGCCCAGGTACTTCTACTCCGGAAAG
  	GTAP		CGGTTCCGCTTCTCCAGGTTCTACTAGC
  			GAATCTCCGTCTGGCACCGCACCA
  LCW0403_066_GFP-	GSTSESPSGTAPGTSP	385	GGTTCTACTAGCGAATCTCCGTCTGGCA	386
  N_B06.ab1	SGESSTAPGTSPSGES		CTGCTCCAGGTACTTCTCCTAGCGGTGA
  	STAP		ATCTTCTACCGCTCCAGGTACTTCCCCTA
  			GCGGCGAATCTTCTACCGCTCCA
  LCW0403_067_GFP-	GSTSESPSGTAPGTST	387	GGTTCTACTAGCGAATCTCCTTCTGGTA	388
  N_C06.ab1	PESGSASPGSTSSTAE		CCGCTCCAGGTACTTCTACCCCTGAAAG
  	SPGP		CGGCTCCGCTTCTCCAGGTTCCACTAGC
  			TCTACCGCTGAATCTCCGGGTCCA
  LCW0403_068_GFP-	GSTSSTAESPGPGSTS	389	GGTTCCACTAGCTCTACTGCTGAATCTC	390
  N_D06.ab1	STAESPGPGSTSESPS		CTGGCCCAGGTTCTACCAGCTCTACCGC
  	GTAP		TGAATCTCCTGGCCCAGGTTCTACCAGC
  			GAATCTCCGTCTGGCACCGCACCA
  LCW0403_069_GFP-	GSTSESPSGTAPGTST	391	GGTTCTACTAGCGAATCCCCGTCTGGTA	392
  N_E06.ab1	PESGSASPGTSTPESG		CCGCACCAGGTACTTCTACCCCGGAAAG
  	SASP		CGGCTCTGCTTCTCCAGGTACTTCTACCC
  			CGGAAAGCGGCTCCGCATCTCCA
  LCW0403_070_GFP-	GSTSESPSGTAPGTST	393	GGTTCTACTAGCGAATCCCCGTCTGGTA	394
  N_F06.ab1	PESGSASPGTSTPESG		CTGCTCCAGGTACTTCTACTCCTGAAAG
  	SASP		CGGTTCCGCTTCTCCAGGTACCTCTACTC
  			CGGAAAGCGGTTCTGCATCTCCA

Example 4Construction of XTEN_AG36 Segments
• • A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AG36. Its segments have the amino acid sequence [X]₃where X is a 12mer peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 43), GSSTPSGATGSP (SEQ ID NO: 44), GSSPSASTGTGP (SEQ ID NO: 45), or GASPGTSSTGSP (SEQ ID NO: 46). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

• (SEQ ID NO: 395)

  	AG1for: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC

  (SEQ ID NO: 396)

  	AG1rev: ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT

  (SEQ ID NO: 397)

  	AG2for: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC

  (SEQ ID NO: 398)

  	AG2rev: ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT

  (SEQ ID NO: 399)

  	AG3for: AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC

  (SEQ ID NO: 400)

  	AG3rev: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA

  (SEQ ID NO: 401)

  	AG4for: AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC

  (SEQ ID NO: 402)

  AG4rev: ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC

• We also annealed the phosphorylatedoligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 139) and thenon-phosphorylated oligonucleotide pr_—3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 140). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0404 showed green fluorescence after induction which shows that the sequence of XTEN_AG36 had been ligated in frame with the GFP gene and most sequences of XTEN_AG36 show good expression.
• We screened 96 isolates from library LCW0404 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 44 clones were identified that contained correct XTEN_AG36 segments. The file names of the nucleotide and amino acid constructs for these segments are listed in Table 12.

• TABLE 12
  DNA and Amino Acid Sequences for 36-mer motifs

  		SEQ		SEQ
  		ID		ID
  File name	Amino acid sequence	NO:	Nucleotide sequence	NO:
  LCW0404_001_GFP-	GASPGTSSTGSPGTPG	403	GGTGCATCCCCGGGCACTAGCTCTACCG	404
  N_A07.ab1	SGTASSSPGSSTPSGA		GTTCTCCAGGTACTCCTGGTAGCGGTAC
  	TGSP		TGCTTCTTCTTCTCCAGGTAGCTCTACTC
  			CTTCTGGTGCTACTGGTTCTCCA
  LCW0404_003_GFP-	GSSTPSGATGSPGSSP	405	GGTAGCTCTACCCCTTCTGGTGCTACCG	406
  N_B07.ab1	SASTGTGPGSSTPSGA		GCTCTCCAGGTTCTAGCCCGTCTGCTTC
  	TGSP		TACCGGTACCGGTCCAGGTAGCTCTACC
  			CCTTCTGGTGCTACTGGTTCTCCA
  LCW0404_006_GFP-	GASPGTSSTGSPGSSP	407	GGTGCATCTCCGGGTACTAGCTCTACCG	408
  N_C07.ab1	SASTGTGPGSSTPSGA		GTTCTCCAGGTTCTAGCCCTTCTGCTTCC
  	TGSP		ACTGGTACCGGCCCAGGTAGCTCTACCC
  			CGTCTGGTGCTACTGGTTCCCCA
  LCW0404_007_GFP-	GTPGSGTASSSPGSST	409	GGTACTCCGGGCAGCGGTACTGCTTCTT	410
  N_D07.ab1	PSGATGSPGASPGTSS		CCTCTCCAGGTAGCTCTACCCCTTCTGG
  	TGSP		TGCAACTGGTTCCCCAGGTGCATCCCCT
  			GGTACTAGCTCTACCGGTTCTCCA
  LCW0404_009_GFP-	GTPGSGTASSSPGASP	411	GGTACCCCTGGCAGCGGTACTGCTTCTT	412
  N_E07.ab1	GTSSTGSPGSRPSAST		CTTCTCCAGGTGCTTCCCCTGGTACCAG
  	GTGP		CTCTACCGGTTCTCCAGGTTCTAGACCT
  			TCTGCATCCACCGGTACTGGTCCA
  LCW0404_011_GFP-	GASPGTSSTGSPGSST	413	GGTGCATCTCCTGGTACCAGCTCTACCG	414
  N_F07.ab1	PSGATGSPGASPGTSS		GTTCTCCAGGTAGCTCTACTCCTTCTGG
  	TGSP		TGCTACTGGCTCTCCAGGTGCTTCCCCG
  			GGTACCAGCTCTACCGGTTCTCCA
  LCW0404_012_GFP-	GTPGSGTASSSPGSST	415	GGTACCCCGGGCAGCGGTACCGCATCTT	416
  N_G07.ab1	PSGATGSPGSSTPSGA		CCTCTCCAGGTAGCTCTACCCCGTCTGG
  	TGSP		TGCTACCGGTTCCCCAGGTAGCTCTACC
  			CCGTCTGGTGCAACCGGCTCCCCA
  LCW0404_014_GFP-	GASPGTSSTGSPGASP	417	GGTGCATCTCCGGGCACTAGCTCTACTG	418
  N_H07.ab1	GTSSTGSPGASPGTSS		GTTCTCCAGGTGCATCCCCTGGCACTAG
  	TGSP		CTCTACTGGTTCTCCAGGTGCTTCTCCTG
  			GTACCAGCTCTACTGGTTCTCCA
  LCW0404_015_GFP-	GSSTPSGATGSPGSSP	419	GGTAGCTCTACTCCGTCTGGTGCAACCG	420
  N_A08.ab1	SASTGTGPGASPGTSS		GCTCCCCAGGTTCTAGCCCGTCTGCTTC
  	TGSP		CACTGGTACTGGCCCAGGTGCTTCCCCG
  			GGCACCAGCTCTACTGGTTCTCCA
  LCW0404_016_GFP-	GSSTPSGATGSPGSST	421	GGTAGCTCTACTCCTTCTGGTGCTACCG	422
  N_B08.ab1	PSGATGSPGTPGSGT		GTTCCCCAGGTAGCTCTACTCCTTCTGG
  	ASSSP		TGCTACTGGTTCCCCAGGTACTCCGGGC
  			AGCGGTACTGCTTCTTCCTCTCCA
  LCW0404_017_GFP-	GSSTPSGATGSPGSST	423	GGTAGCTCTACTCCGTCTGGTGCAACCG	424
  N_C08.ab1	PSGATGSPGASPGTSS		GTTCCCCAGGTAGCTCTACTCCTTCTGG
  	TGSP		TGCTACTGGCTCCCCAGGTGCATCCCCT
  			GGCACCAGCTCTACCGGTTCTCCA
  LCW0404_018_GFP-	GTPGSGTASSSPGSSP	425	GGTACTCCTGGTAGCGGTACCGCATCTT	426
  N_D08.ab1	SASTGTGPGSSTPSGA		CCTCTCCAGGTTCTAGCCCTTCTGCATCT
  	TGSP		ACCGGTACCGGTCCAGGTAGCTCTACTC
  			CTTCTGGTGCTACTGGCTCTCCA
  LCW0404_023_GFP-	GASPGTSSTGSPGSSP	427	GGTGCTTCCCCGGGCACTAGCTCTACCG	428
  N_F08.ab1	SASTGTGPGTPGSGT		GTTCTCCAGGTTCTAGCCCTTCTGCATCT
  	ASSSP		ACTGGTACTGGCCCAGGTACTCCGGGCA
  			GCGGTACTGCTTCTTCCTCTCCA
  LCW0404_025_GFP-	GSSTPSGATGSPGSST	429	GGTAGCTCTACTCCGTCTGGTGCTACCG	430
  N_G08.ab1	PSGATGSPGASPGTSS		GCTCTCCAGGTAGCTCTACCCCTTCTGG
  	TGSP		TGCAACCGGCTCCCCAGGTGCTTCTCCG
  			GGTACCAGCTCTACTGGTTCTCCA
  LCW0404_029_GFP-	GTPGSGTASSSPGSST	431	GGTACCCCTGGCAGCGGTACCGCTTCTT	432
  N_A09.ab1	PSGATGSPGSSPSAST		CCTCTCCAGGTAGCTCTACCCCGTCTGG
  	GTGP		TGCTACTGGCTCTCCAGGTTCTAGCCCG
  			TCTGCATCTACCGGTACCGGCCCA
  LCW0404_030_GFP-	GSSTPSGATGSPGTPG	433	GGTAGCTCTACTCCTTCTGGTGCAACCG	434
  N_B09.ab1	SGTASSSPGTPGSGTA		GCTCCCCAGGTACCCCGGGCAGCGGTA
  	SSSP		CCGCATCTTCCTCTCCAGGTACTCCGGG
  			TAGCGGTACTGCTTCTTCTTCTCCA
  LCW0404_031_GFP-	GTPGSGTASSSPGSST	435	GGTACCCCGGGTAGCGGTACTGCTTCTT	436
  N_C09.ab1	PSGATGSPGASPGTSS		CCTCTCCAGGTAGCTCTACCCCTTCTGG
  	TGSP		TGCAACCGGCTCTCCAGGTGCTTCTCCG
  			GGCACCAGCTCTACCGGTTCTCCA
  LCW0404_034_GFP-	GSSTPSGATGSPGSST	437	GGTAGCTCTACCCCGTCTGGTGCTACCG	438
  N_D09.ab1	PSGATGSPGASPGTSS		GCTCTCCAGGTAGCTCTACCCCGTCTGG
  	TGSP		TGCAACCGGCTCCCCAGGTGCATCCCCG
  			GGTACTAGCTCTACCGGTTCTCCA
  LCW0404_035_GFP-	GASPGTSSTGSPGTPG	439	GGTGCTTCTCCGGGCACCAGCTCTACTG	440
  N_E09.ab1	SGTASSSPGSSTPSGA		GTTCTCCAGGTACCCCGGGCAGCGGTAC
  	TGSP		CGCATCTTCTTCTCCAGGTAGCTCTACT
  			CCTTCTGGTGCAACTGGTTCTCCA
  LCW0404_036_GFP-	GSSPSASTGTGPGSST	441	GGTTCTAGCCCGTCTGCTTCCACCGGTA	442
  N_F09.ab1	PSGATGSPGTPGSGT		CTGGCCCAGGTAGCTCTACCCCGTCTGG
  	ASSSP		TGCAACTGGTTCCCCAGGTACCCCTGGT
  			AGCGGTACCGCTTCTTCTTCTCCA
  LCW0404_037_GFP-	GASPGTSSTGSPGSSP	443	GGTGCTTCTCCGGGCACCAGCTCTACTG	444
  N_G09.ab1	SASTGTGPGSSTPSGA		GTTCTCCAGGTTCTAGCCCTTCTGCATC
  	TGSP		CACCGGTACCGGTCCAGGTAGCTCTACC
  			CCTTCTGGTGCAACCGGCTCTCCA
  LCW0404_040_GFP-	GASPGTSSTGSPGSST	445	GGTGCATCCCCGGGCACCAGCTCTACCG	446
  N_H09.ab1	PSGATGSPGSSTPSGA		GTTCTCCAGGTAGCTCTACCCCGTCTGG
  	TGSP		TGCTACCGGCTCTCCAGGTAGCTCTACC
  			CCGTCTGGTGCTACTGGCTCTCCA
  LCW0404_041_GFP-	GTPGSGTASSSPGSST	447	GGTACCCCTGGTAGCGGTACTGCTTCTT	448
  N_A10.ab1	PSGATGSPGTPGSGT		CCTCTCCAGGTAGCTCTACTCCGTCTGG
  	ASSSP		TGCTACCGGTTCTCCAGGTACCCCGGGT
  			AGCGGTACCGCATCTTCTTCTCCA
  LCW0404_043_GFP-	GSSPSASTGTGPGSST	449	GGTTCTAGCCCTTCTGCTTCCACCGGTA	450
  N_C10.ab1	PSGATGSPGSSTPSGA		CTGGCCCAGGTAGCTCTACCCCTTCTGG
  	TGSP		TGCTACCGGCTCCCCAGGTAGCTCTACT
  			CCTTCTGGTGCAACTGGCTCTCCA
  LCW0404_045_GFP-	GASPGTSSTGSPGSSP	451	GGTGCTTCTCCTGGCACCAGCTCTACTG	452
  N_D10.ab1	SASTGTGPGSSPSAST		GTTCTCCAGGTTCTAGCCCTTCTGCTTCT
  	GTGP		ACCGGTACTGGTCCAGGTTCTAGCCCTT
  			CTGCATCCACTGGTACTGGTCCA
  LCW0404_047_GFP-	GTPGSGTASSSPGASP	453	GGTACTCCTGGCAGCGGTACCGCTTCTT	454
  N_F10.ab1	GTSSTGSPGASPGTSS		CTTCTCCAGGTGCTTCTCCTGGTACTAG
  	TGSP		CTCTACTGGTTCTCCAGGTGCTTCTCCG
  			GGCACTAGCTCTACTGGTTCTCCA
  LCW0404_048_GFP-	GSSTPSGATGSPGASP	455	GGTAGCTCTACCCCGTCTGGTGCTACCG	456
  N_G10.ab1	GTSSTGSPGSSTPSGA		GTTCCCCAGGTGCTTCTCCTGGTACTAG
  	TGSP		CTCTACCGGTTCTCCAGGTAGCTCTACC
  			CCGTCTGGTGCTACTGGCTCTCCA
  LCW0404_049_GFP-	GSSTPSGATGSPGTPG	457	GGTAGCTCTACCCCGTCTGGTGCTACTG	458
  N_H10.ab1	SGTASSSPGSSTPSGA		GTTCTCCAGGTACTCCGGGCAGCGGTAC
  	TGSP		TGCTTCTTCCTCTCCAGGTAGCTCTACCC
  			CTTCTGGTGCTACTGGCTCTCCA
  LCW0404_050_GFP-	GASPGTSSTGSPGSSP	459	GGTGCATCTCCTGGTACCAGCTCTACTG	460
  N_A11.ab1	SASTGTGPGSSTPSGA		GTTCTCCAGGTTCTAGCCCTTCTGCTTCT
  	TGSP		ACCGGTACCGGTCCAGGTAGCTCTACTC
  			CTTCTGGTGCTACCGGTTCTCCA
  LCW0404_051_GFP-	GSSTPSGATGSPGSST	461	GGTAGCTCTACCCCGTCTGGTGCTACTG	462
  N_B11.ab1	PSGATGSPGSSTPSGA		GCTCTCCAGGTAGCTCTACTCCTTCTGG
  	TGSP		TGCTACTGGTTCCCCAGGTAGCTCTACC
  			CCGTCTGGTGCAACTGGCTCTCCA
  LCW0404_052_GFP-	GASPGTSSTGSPGTPG	463	GGTGCATCCCCGGGTACCAGCTCTACCG	464
  N_C11.ab1	SGTASSSPGASPGTSS		GTTCTCCAGGTACTCCTGGCAGCGGTAC
  	TGSP		TGCATCTTCCTCTCCAGGTGCTTCTCCG
  			GGCACCAGCTCTACTGGTTCTCCA
  LCW0404_053_GFP-	GSSTPSGATGSPGSSP	465	GGTAGCTCTACTCCTTCTGGTGCAACTG	466
  N_D11.ab1	SASTGTGPGASPGTSS		GTTCTCCAGGTTCTAGCCCGTCTGCATC
  	TGSP		CACTGGTACCGGTCCAGGTGCTTCCCCT
  			GGCACCAGCTCTACCGGTTCTCCA
  LCW0404_057_GFP-	GASPGTSSTGSPGSST	467	GGTGCATCTCCTGGTACTAGCTCTACTG	468
  N_E11.ab1	PSGATGSPGSSPSAST		GTTCTCCAGGTAGCTCTACTCCGTCTGG
  	GTGP		TGCAACCGGCTCTCCAGGTTCTAGCCCT
  			TCTGCATCTACCGGTACTGGTCCA
  LCW0404_060_GFP-	GTPGSGTASSSPGSST	469	GGTACTCCTGGCAGCGGTACCGCATCTT	470
  N_F11.ab1	PSGATGSPGASPGTSS		CCTCTCCAGGTAGCTCTACTCCGTCTGG
  	TGSP		TGCAACTGGTTCCCCAGGTGCTTCTCCG
  			GGTACCAGCTCTACCGGTTCTCCA
  LCW0404_062_GFP-	GSSTPSGATGSPGTPG	471	GGTAGCTCTACCCCGTCTGGTGCAACCG	472
  N_G11.ab1	SGTASSSPGSSTPSGA		GCTCCCCAGGTACTCCTGGTAGCGGTAC
  	TGSP		CGCTTCTTCTTCTCCAGGTAGCTCTACTC
  			CGTCTGGTGCTACCGGCTCCCCA
  LCW0404_066_GFP-	GSSPSASTGTGPGSSP	473	GGTTCTAGCCCTTCTGCATCCACCGGTA	474
  N_H11.ab1	SASTGTGPGASPGTSS		CCGGCCCAGGTTCTAGCCCGTCTGCTTC
  	TGSP		TACCGGTACTGGTCCAGGTGCTTCTCCG
  			GGTACTAGCTCTACTGGTTCTCCA
  LCW0404_067_GFP-	GTPGSGTASSSPGSST	475	GGTACCCCGGGTAGCGGTACCGCTTCTT	476
  N_A12.ab1	PSGATGSPGSNPSAST		CTTCTCCAGGTAGCTCTACTCCGTCTGG
  	GTGP		TGCTACCGGCTCTCCAGGTTCTAACCCT
  			TCTGCATCCACCGGTACCGGCCCA
  LCW0404_068_GFP-	GSSPSASTGTGPGSST	477	GGTTCTAGCCCTTCTGCATCTACTGGTA	478
  N_B12.ab1	PSGATGSPGASPGTSS		CTGGCCCAGGTAGCTCTACTCCTTCTGG
  	TGSP		TGCTACCGGCTCTCCAGGTGCTTCTCCG
  			GGTACTAGCTCTACCGGTTCTCCA
  LCW0404_069_GFP-	GSSTPSGATGSPGASP	479	GGTAGCTCTACCCCTTCTGGTGCAACCG	480
  N_C12.ab1	GTSSTGSPGTPGSGTA		GCTCTCCAGGTGCATCCCCGGGTACCAG
  	SSSP		CTCTACCGGTTCTCCAGGTACTCCGGGT
  			AGCGGTACCGCTTCTTCCTCTCCA
  LCW0404_070_GFP-	GSSTPSGATGSPGSST	481	GGTAGCTCTACTCCGTCTGGTGCAACCG	482
  N_D12.ab1	PSGATGSPGSSTPSGA		GTTCCCCAGGTAGCTCTACCCCTTCTGG
  	TGSP		TGCAACCGGCTCCCCAGGTAGCTCTACC
  			CCTTCTGGTGCAACTGGCTCTCCA
  LCW0404_073_GFP-	GASPGTSSTGSPGTPG	483	GGTGCTTCTCCTGGCACTAGCTCTACCG	484
  N_E12.ab1	SGTASSSPGSSTPSGA		GTTCTCCAGGTACCCCTGGTAGCGGTAC
  	TGSP		CGCATCTTCCTCTCCAGGTAGCTCTACT
  			CCTTCTGGTGCTACTGGTTCCCCA
  LCW0404_075_GFP-	GSSTPSGATGSPGSSP	485	GGTAGCTCTACCCCGTCTGGTGCTACTG	486
  N_F12.ab1	SASTGTGPGSSPSAST		GCTCCCCAGGTTCTAGCCCTTCTGCATC
  	GTGP		CACCGGTACCGGTCCAGGTTCTAGCCCG
  			TCTGCATCTACTGGTACTGGTCCA
  LCW0404_080_GFP-	GASPGTSSTGSPGSSP	487	GGTGCTTCCCCGGGCACCAGCTCTACTG	488
  N_G12.ab1	SASTGTGPGSSPSAST		GTTCTCCAGGTTCTAGCCCGTCTGCTTCT
  	GTGP		ACTGGTACTGGTCCAGGTTCTAGCCCTT
  			CTGCTTCCACTGGTACTGGTCCA
  LCW0404_081_GFP-	GASPGTSSTGSPGSSP	489	GGTGCTTCCCCGGGTACCAGCTCTACCG	490
  N_H12.ab1	SASTGTGPGTPGSGT		GTTCTCCAGGTTCTAGCCCTTCTGCTTCT
  	ASSSP		ACCGGTACCGGTCCAGGTACCCCTGGCA
  			GCGGTACCGCATCTTCCTCTCCA

Example 5Construction of XTEN_AE864
• XTEN_AE864 was constructed from serial dimerization of XTEN_AE36 to AE72, 144, 288, 576 and 864. A collection of XTEN_AE72 segments was constructed from 37 different segments of XTEN_AE36. Cultures ofE. coliharboring all 37 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment as the insert. The same plasmid pool was digested with BbsI/NcoI to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AE72.
• This library of XTEN_AE72 segments was designated LCW0406. All clones from LCW0406 were combined and dimerized again using the same process as described above yielding library LCW0410 of XTEN_AE144. All clones from LCW0410 were combined and dimerized again using the same process as described above yielding library LCW0414 of XTEN_AE288. Two isolates LCW0414.001 and LCW0414.002 were randomly picked from the library and sequenced to verify the identities. All clones from LCW0414 were combined and dimerized again using the same process as described above yielding library LCW0418 of XTEN_AE576. We screened 96 isolates from library LCW0418 for high level of GFP fluorescence. 8 isolates with right sizes of inserts by PCR and strong fluorescence were sequenced and 2 isolates (LCW0418.018 and LCW0418.052) were chosen for future use based on sequencing and expression data.
• The specific clone pCW0432 of XTEN_AE864 was constructed by combining LCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288 using the same dimerization process as described above.
Example 6Construction of XTEN_AM144
• A collection of XTEN_AM144 segments was constructed starting from 37 different segments of XTEN_AE36, 44 segments of XTEN_AF36, and 44 segments of XTEN_AG36.
• Cultures ofE. coliharboring all 125 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment as the insert. The same plasmid pool was digested with BbsI/NcoI to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AM72.
• This library of XTEN_AM72 segments was designated LCW0461. All clones from LCW0461 were combined and dimerized again using the same process as described above yielding library LCW0462. 1512 Isolates from library LCW0462 were screened for protein expression. Individual colonies were transferred into 96 well plates and cultured overnight as starter cultures. These starter cultures were diluted into fresh autoinduction medium and cultured for 20-30 h. Expression was measured using a fluorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showed high level expression and were submitted to DNA sequencing. Most clones in library LCW0462 showed good expression and similar physicochemical properties suggesting that most combinations of XTEN_AM36 segments yield useful XTEN sequences. 30 isolates from LCW0462 were chosen as a preferred collection of XTEN_AM144 segments for the construction of multifunctional proteins that contain multiple XTEN segments. The file names of the nucleotide and amino acid constructs for these segments are listed in Table 13.

• TABLE 13
  DNA and amino acid sequences for AM144 segments

  		SEQ		SEQ
  		ID		ID
  Clone	Sequence Trimmed	NO:	Protein Sequence	NO:
  LCW462_r1	GGTACCCCGGGCAGCGGTACCGCATCTT	491	GTPGSGTASSSPGSSTPS	492
  	CCTCTCCAGGTAGCTCTACCCCGTCTGGT		GATGSPGSSTPSGATGSP
  	GCTACCGGTTCCCCAGGTAGCTCTACCCC		GSPAGSPTSTEEGTSESA
  	GTCTGGTGCAACCGGCTCCCCAGGTAGC		TPESGPGTSTEPSEGSAP
  	CCGGCTGGCTCTCCTACCTCTACTGAGGA		GSSPSASTGTGPGSSPSA
  	AGGTACTTCTGAAAGCGCTACTCCTGAG		STGTGPGASPGTSSTGSP
  	TCTGGTCCAGGTACCTCTACTGAACCGTC		GTSTEPSEGSAPGTSTEP
  	CGAAGGTAGCGCTCCAGGTTCTAGCCCT		SEGSAPGSEPATSGSETP
  	TCTGCATCCACCGGTACCGGCCCAGGTTC
  	TAGCCCGTCTGCTTCTACCGGTACTGGTC
  	CAGGTGCTTCTCCGGGTACTAGCTCTACT
  	GGTTCTCCAGGTACCTCTACCGAACCGTC
  	CGAGGGTAGCGCACCAGGTACCTCTACT
  	GAACCGTCTGAGGGTAGCGCTCCAGGTA
  	GCGAACCGGCAACCTCCGGTTCTGAAAC
  	TCCA
  LCW462_r5	GGTTCTACCAGCGAATCCCCTTCTGGCAC	493	GSTSESPSGTAPGSTSESP	494
  	TGCACCAGGTTCTACTAGCGAATCCCCTT		SGTAPGTSPSGESSTAPG
  	CTGGTACCGCACCAGGTACTTCTCCGAG		TSTEPSEGSAPGTSTEPSE
  	CGGCGAATCTTCTACTGCTCCAGGTACCT		GSAPGTSESATPESGPGA
  	CTACTGAACCTTCCGAAGGCAGCGCTCC		SPGTSSTGSPGSSTPSGA
  	AGGTACCTCTACCGAACCGTCCGAGGGC		TGSPGASPGTSSTGSPGS
  	AGCGCACCAGGTACTTCTGAAAGCGCAA		TSESPSGTAPGSTSESPSG
  	CCCCTGAATCCGGTCCAGGTGCATCTCCT		TAPGTSTPESGSASP
  	GGTACCAGCTCTACCGGTTCTCCAGGTA
  	GCTCTACTCCTTCTGGTGCTACTGGCTCT
  	CCAGGTGCTTCCCCGGGTACCAGCTCTAC
  	CGGTTCTCCAGGTTCTACTAGCGAATCTC
  	CTTCTGGCACTGCACCAGGTTCTACCAGC
  	GAATCTCCGTCTGGCACTGCACCAGGTA
  	CCTCTACCCCTGAAAGCGGTTCCGCTTCT
  	CCA
  LCW462_r9	GGTACTTCTACCGAACCTTCCGAGGGCA	495	GTSTEPSEGSAPGTSESA	496
  	GCGCACCAGGTACTTCTGAAAGCGCTAC		TPESGPGTSESATPESGP
  	CCCTGAGTCCGGCCCAGGTACTTCTGAA		GTSTEPSEGSAPGTSESA
  	AGCGCTACTCCTGAATCCGGTCCAGGTA		TPESGPGTSTEPSEGSAP
  	CCTCTACTGAACCTTCTGAGGGCAGCGCT		GTSTEPSEGSAPGSEPAT
  	CCAGGTACTTCTGAAAGCGCTACCCCGG		SGSETPGSPAGSPTSTEE
  	AGTCCGGTCCAGGTACTTCTACTGAACC		GASPGTSSTGSPGSSPSA
  	GTCCGAAGGTAGCGCACCAGGTACTTCT		STGTGPGSSPSASTGTGP
  	ACTGAACCTTCCGAAGGTAGCGCTCCAG
  	GTAGCGAACCTGCTACTTCTGGTTCTGAA
  	ACCCCAGGTAGCCCGGCTGGCTCTCCGA
  	CCTCCACCGAGGAAGGTGCTTCTCCTGG
  	CACCAGCTCTACTGGTTCTCCAGGTTCTA
  	GCCCTTCTGCTTCTACCGGTACTGGTCCA
  	GGTTCTAGCCCTTCTGCATCCACTGGTAC
  	TGGTCCA
  LCW462_r10	GGTAGCGAACCGGCAACCTCTGGCTCTG	497	GSEPATSGSETPGTSESA	498
  	AAACCCCAGGTACCTCTGAAAGCGCTAC		TPESGPGTSESATPESGP
  	TCCGGAATCTGGTCCAGGTACTTCTGAA		GSTSESPSGTAPGSTSESP
  	AGCGCTACTCCGGAATCCGGTCCAGGTT		SGTAPGTSPSGESSTAPG
  	CTACCAGCGAATCTCCTTCTGGCACCGCT		ASPGTSSTGSPGSSPSAS
  	CCAGGTTCTACTAGCGAATCCCCGTCTGG		TGTGPGSSTPSGATGSPG
  	TACCGCACCAGGTACTTCTCCTAGCGGC		SSTPSGATGSPGSSTPSG
  	GAATCTTCTACCGCACCAGGTGCATCTCC		ATGSPGASPGTSSTGSP
  	GGGTACTAGCTCTACCGGTTCTCCAGGTT
  	CTAGCCCTTCTGCTTCCACTGGTACCGGC
  	CCAGGTAGCTCTACCCCGTCTGGTGCTAC
  	TGGTTCCCCAGGTAGCTCTACTCCGTCTG
  	GTGCAACCGGTTCCCCAGGTAGCTCTACT
  	CCTTCTGGTGCTACTGGCTCCCCAGGTGC
  	ATCCCCTGGCACCAGCTCTACCGGTTCTC
  	CA
  LCW462_r15	GGTGCTTCTCCGGGCACCAGCTCTACTGG	499	GASPGTSSTGSPGSSPSA	500
  	TTCTCCAGGTTCTAGCCCTTCTGCATCCA		STGTGPGSSTPSGATGSP
  	CCGGTACCGGTCCAGGTAGCTCTACCCCT		GTSESATPESGPGSEPAT
  	TCTGGTGCAACCGGCTCTCCAGGTACTTC		SGSETPGSEPATSGSETP
  	TGAAAGCGCTACCCCGGAATCTGGCCCA		GTSESATPESGPGTSTEP
  	GGTAGCGAACCGGCTACTTCTGGTTCTG		SEGSAPGTSTEPSEGSAP
  	AAACCCCAGGTAGCGAACCGGCTACCTC		GTSTEPSEGSAPGTSTEP
  	CGGTTCTGAAACTCCAGGTACTTCTGAA		SEGSAPGSEPATSGSETP
  	AGCGCTACTCCGGAGTCCGGTCCAGGTA
  	CCTCTACCGAACCGTCCGAAGGCAGCGC
  	TCCAGGTACTTCTACTGAACCTTCTGAGG
  	GTAGCGCTCCAGGTACCTCTACCGAACC
  	GTCCGAGGGTAGCGCACCAGGTACCTCT
  	ACTGAACCGTCTGAGGGTAGCGCTCCAG
  	GTAGCGAACCGGCAACCTCCGGTTCTGA
  	AACTCCA
  LCW462_r16	GGTACCTCTACCGAACCTTCCGAAGGTA	501	GTSTEPSEGSAPGSPAGS	502
  	GCGCTCCAGGTAGCCCGGCAGGTTCTCC		PTSTEEGTSTEPSEGSAP
  	TACTTCCACTGAGGAAGGTACTTCTACCG		GTSESATPESGPGSEPAT
  	AACCTTCTGAGGGTAGCGCACCAGGTAC		SGSETPGTSESATPESGP
  	CTCTGAAAGCGCAACTCCTGAGTCTGGC		GSPAGSPTSTEEGTSESA
  	CCAGGTAGCGAACCTGCTACCTCCGGCT		TPESGPGTSTEPSEGSAP
  	CTGAGACTCCAGGTACCTCTGAAAGCGC		GSEPATSGSETPGTSTEP
  	AACCCCGGAATCTGGTCCAGGTAGCCCG		SEGSAPGSEPATSGSETP
  	GCTGGCTCTCCTACCTCTACTGAGGAAG
  	GTACTTCTGAAAGCGCTACTCCTGAGTCT
  	GGTCCAGGTACCTCTACTGAACCGTCCG
  	AAGGTAGCGCTCCAGGTAGCGAACCTGC
  	TACTTCTGGTTCTGAAACTCCAGGTACTT
  	CTACCGAACCGTCCGAGGGTAGCGCTCC
  	AGGTAGCGAACCTGCTACTTCTGGTTCTG
  	AAACTCCA
  LCW462_r20	GGTACTTCTACCGAACCGTCCGAAGGCA	503	GTSTEPSEGSAPGTSTEP	504
  	GCGCTCCAGGTACCTCTACTGAACCTTCC		SEGSAPGTSTEPSEGSAP
  	GAGGGCAGCGCTCCAGGTACCTCTACCG		GTSTEPSEGSAPGTSTEP
  	AACCTTCTGAAGGTAGCGCACCAGGTAC		SEGSAPGTSTEPSEGSAP
  	TTCTACCGAACCGTCCGAAGGCAGCGCT		GTSTEPSEGSAPGTSESA
  	CCAGGTACCTCTACTGAACCTTCCGAGG		TPESGPGTSESATPESGP
  	GCAGCGCTCCAGGTACCTCTACCGAACC		GTSTEPSEGSAPGSEPAT
  	TTCTGAAGGTAGCGCACCAGGTACTTCT		SGSETPGSPAGSPTSTEE
  	ACCGAACCTTCCGAGGGCAGCGCACCAG
  	GTACTTCTGAAAGCGCTACCCCTGAGTCC
  	GGCCCAGGTACTTCTGAAAGCGCTACTC
  	CTGAATCCGGTCCAGGTACTTCTACTGAA
  	CCTTCCGAAGGTAGCGCTCCAGGTAGCG
  	AACCTGCTACTTCTGGTTCTGAAACCCCA
  	GGTAGCCCGGCTGGCTCTCCGACCTCCA
  	CCGAGGAA
  LCW462_r23	GGTACTTCTACCGAACCGTCCGAGGGCA	505	GTSTEPSEGSAPGTSTEP	506
  	GCGCTCCAGGTACTTCTACTGAACCTTCT		SEGSAPGTSTEPSEGSAP
  	GAAGGCAGCGCTCCAGGTACTTCTACTG		GSTSESPSGTAPGSTSESP
  	AACCTTCCGAAGGTAGCGCACCAGGTTC		SGTAPGTSTPESGSASPG
  	TACCAGCGAATCCCCTTCTGGTACTGCTC		SEPATSGSETPGTSESAT
  	CAGGTTCTACCAGCGAATCCCCTTCTGGC		PESGPGTSTEPSEGSAPG
  	ACCGCACCAGGTACTTCTACCCCTGAAA		TSTEPSEGSAPGTSESAT
  	GCGGCTCCGCTTCTCCAGGTAGCGAACC		PESGPGTSESATPESGP
  	TGCAACCTCTGGCTCTGAAACCCCAGGT
  	ACCTCTGAAAGCGCTACTCCTGAATCTG
  	GCCCAGGTACTTCTACTGAACCGTCCGA
  	GGGCAGCGCACCAGGTACTTCTACTGAA
  	CCGTCTGAAGGTAGCGCACCAGGTACTT
  	CTGAAAGCGCAACCCCGGAATCCGGCCC
  	AGGTACCTCTGAAAGCGCAACCCCGGAG
  	TCCGGCCCA
  LCW462_r24	GGTAGCTCTACCCCTTCTGGTGCTACCGG	507	GSSTPSGATGSPGSSPSA	508
  	CTCTCCAGGTTCTAGCCCGTCTGCTTCTA		STGTGPGSSTPSGATGSP
  	CCGGTACCGGTCCAGGTAGCTCTACCCCT		GSPAGSPTSTEEGSPAGS
  	TCTGGTGCTACTGGTTCTCCAGGTAGCCC		PTSTEEGTSTEPSEGSAP
  	TGCTGGCTCTCCGACTTCTACTGAGGAAG		GASPGTSSTGSPGSSPSA
  	GTAGCCCGGCTGGTTCTCCGACTTCTACT		STGTGPGTPGSGTASSSP
  	GAGGAAGGTACTTCTACCGAACCTTCCG		GSTSSTAESPGPGTSPSG
  	AAGGTAGCGCTCCAGGTGCTTCCCCGGG		ESSTAPGTSTPESGSASP
  	CACTAGCTCTACCGGTTCTCCAGGTTCTA
  	GCCCTTCTGCATCTACTGGTACTGGCCCA
  	GGTACTCCGGGCAGCGGTACTGCTTCTTC
  	CTCTCCAGGTTCTACTAGCTCTACTGCTG
  	AATCTCCTGGCCCAGGTACTTCTCCTAGC
  	GGTGAATCTTCTACCGCTCCAGGTACCTC
  	TACTCCGGAAAGCGGTTCTGCATCTCCA
  LCW462_r27	GGTACCTCTACTGAACCTTCTGAGGGCA	509	GTSTEPSEGSAPGTSESA	510
  	GCGCTCCAGGTACTTCTGAAAGCGCTAC		TPESGPGTSTEPSEGSAP
  	CCCGGAGTCCGGTCCAGGTACTTCTACTG		GTSTEPSEGSAPGTSESA
  	AACCGTCCGAAGGTAGCGCACCAGGTAC		TPESGPGTSESATPESGP
  	TTCTACTGAACCGTCTGAAGGTAGCGCA		GTPGSGTASSSPGASPGT
  	CCAGGTACTTCTGAAAGCGCAACCCCGG		SSTGSPGASPGTSSTGSP
  	AATCCGGCCCAGGTACCTCTGAAAGCGC		GSPAGSPTSTEEGSPAGS
  	AACCCCGGAGTCCGGCCCAGGTACTCCT		PTSTEEGTSTEPSEGSAP
  	GGCAGCGGTACCGCTTCTTCTTCTCCAGG
  	TGCTTCTCCTGGTACTAGCTCTACTGGTT
  	CTCCAGGTGCTTCTCCGGGCACTAGCTCT
  	ACTGGTTCTCCAGGTAGCCCTGCTGGCTC
  	TCCGACTTCTACTGAGGAAGGTAGCCCG
  	GCTGGTTCTCCGACTTCTACTGAGGAAG
  	GTACTTCTACCGAACCTTCCGAAGGTAG
  	CGCTCCA
  LCW462_r28	GGTAGCCCAGCAGGCTCTCCGACTTCCA	511	GSPAGSPTSTEEGTSTEP	512
  	CTGAGGAAGGTACTTCTACTGAACCTTCC		SEGSAPGTSTEPSEGSAP
  	GAAGGCAGCGCACCAGGTACCTCTACTG		GTSTEPSEGSAPGTSESA
  	AACCTTCTGAGGGCAGCGCTCCAGGTAC		TPESGPGTSESATPESGP
  	CTCTACCGAACCGTCTGAAGGTAGCGCA		GTPGSGTASSSPGSSTPS
  	CCAGGTACCTCTGAAAGCGCAACTCCTG		GATGSPGASPGTSSTGSP
  	AGTCCGGTCCAGGTACTTCTGAAAGCGC		GTSTEPSEGSAPGTSESA
  	AACCCCGGAGTCTGGCCCAGGTACCCCG		TPESGPGTSTEPSEGSAP
  	GGTAGCGGTACTGCTTCTTCCTCTCCAGG
  	TAGCTCTACCCCTTCTGGTGCAACCGGCT
  	CTCCAGGTGCTTCTCCGGGCACCAGCTCT
  	ACCGGTTCTCCAGGTACCTCTACTGAACC
  	TTCTGAGGGCAGCGCTCCAGGTACTTCTG
  	AAAGCGCTACCCCGGAGTCCGGTCCAGG
  	TACTTCTACTGAACCGTCCGAAGGTAGC
  	GCACCA
  LCW462_r38	GGTAGCGAACCGGCAACCTCCGGCTCTG	513	GSEPATSGSETPGTSESA	514
  	AAACTCCAGGTACTTCTGAAAGCGCTAC		TPESGPGSEPATSGSETP
  	TCCGGAATCCGGCCCAGGTAGCGAACCG		GSSTPSGATGSPGTPGSG
  	GCTACTTCCGGCTCTGAAACCCCAGGTA		TASSSPGSSTPSGATGSP
  	GCTCTACCCCGTCTGGTGCAACCGGCTCC		GASPGTSSTGSPGSSTPS
  	CCAGGTACTCCTGGTAGCGGTACCGCTTC		GATGSPGASPGTSSTGSP
  	TTCTTCTCCAGGTAGCTCTACTCCGTCTG		GSEPATSGSETPGTSTEP
  	GTGCTACCGGCTCCCCAGGTGCATCTCCT		SEGSAPGSEPATSGSETP
  	GGTACCAGCTCTACCGGTTCTCCAGGTA
  	GCTCTACTCCTTCTGGTGCTACTGGCTCT
  	CCAGGTGCTTCCCCGGGTACCAGCTCTAC
  	CGGTTCTCCAGGTAGCGAACCTGCTACTT
  	CTGGTTCTGAAACTCCAGGTACTTCTACC
  	GAACCGTCCGAGGGTAGCGCTCCAGGTA
  	GCGAACCTGCTACTTCTGGTTCTGAAACT
  	CCA
  LCW462_r39	GGTACCTCTACTGAACCTTCCGAAGGCA	515	GTSTEPSEGSAPGTSTEP	516
  	GCGCTCCAGGTACCTCTACCGAACCGTC		SEGSAPGTSESATPESGP
  	CGAGGGCAGCGCACCAGGTACTTCTGAA		GSPAGSPTSTEEGSPAGS
  	AGCGCAACCCCTGAATCCGGTCCAGGTA		PTSTEEGTSTEPSEGSAP
  	GCCCTGCTGGCTCTCCGACTTCTACTGAG		GSPAGSPTSTEEGTSTEP
  	GAAGGTAGCCCGGCTGGTTCTCCGACTT		SEGSAPGTSTEPSEGSAP
  	CTACTGAGGAAGGTACTTCTACCGAACC		GASPGTSSTGSPGSSPSA
  	TTCCGAAGGTAGCGCTCCAGGTAGCCCG		STGTGPGSSPSASTGTGP
  	GCTGGTTCTCCGACTTCCACCGAGGAAG
  	GTACCTCTACTGAACCTTCTGAGGGTAGC
  	GCTCCAGGTACCTCTACTGAACCTTCCGA
  	AGGCAGCGCTCCAGGTGCTTCCCCGGGC
  	ACCAGCTCTACTGGTTCTCCAGGTTCTAG
  	CCCGTCTGCTTCTACTGGTACTGGTCCAG
  	GTTCTAGCCCTTCTGCTTCCACTGGTACT
  	GGTCCA
  LCW462_r41	GGTAGCTCTACCCCGTCTGGTGCTACCGG	517	GSSTPSGATGSPGASPGT	518
  	TTCCCCAGGTGCTTCTCCTGGTACTAGCT		SSTGSPGSSTPSGATGSP
  	CTACCGGTTCTCCAGGTAGCTCTACCCCG		GSPAGSPTSTEEGTSESA
  	TCTGGTGCTACTGGCTCTCCAGGTAGCCC		TPESGPGSEPATSGSETP
  	TGCTGGCTCTCCAACCTCCACCGAAGAA		GASPGTSSTGSPGSSTPS
  	GGTACCTCTGAAAGCGCAACCCCTGAAT		GATGSPGSSPSASTGTGP
  	CCGGCCCAGGTAGCGAACCGGCAACCTC		GSTSESPSGTAPGSTSESP
  	CGGTTCTGAAACCCCAGGTGCATCTCCTG		SGTAPGTSTPESGSASP
  	GTACTAGCTCTACTGGTTCTCCAGGTAGC
  	TCTACTCCGTCTGGTGCAACCGGCTCTCC
  	AGGTTCTAGCCCTTCTGCATCTACCGGTA
  	CTGGTCCAGGTTCTACCAGCGAATCCCCT
  	TCTGGTACTGCTCCAGGTTCTACCAGCGA
  	ATCCCCTTCTGGCACCGCACCAGGTACTT
  	CTACCCCTGAAAGCGGCTCCGCTTCTCCA
  LCW462_r42	GGTTCTACCAGCGAATCTCCTTCTGGCAC	519	GSTSESPSGTAPGSTSESP	520
  	CGCTCCAGGTTCTACTAGCGAATCCCCGT		SGTAPGTSPSGESSTAPG
  	CTGGTACCGCACCAGGTACTTCTCCTAGC		TSESATPESGPGTSTEPSE
  	GGCGAATCTTCTACCGCACCAGGTACCT		GSAPGTSTEPSEGSAPGT
  	CTGAAAGCGCTACTCCGGAGTCTGGCCC		STEPSEGSAPGTSESATP
  	AGGTACCTCTACTGAACCGTCTGAGGGT		ESGPGTSTEPSEGSAPGS
  	AGCGCTCCAGGTACTTCTACTGAACCGTC		STPSGATGSPGASPGTSS
  	CGAAGGTAGCGCACCAGGTACCTCTACT		TGSPGSSTPSGATGSP
  	GAACCTTCTGAGGGCAGCGCTCCAGGTA
  	CTTCTGAAAGCGCTACCCCGGAGTCCGG
  	TCCAGGTACTTCTACTGAACCGTCCGAA
  	GGTAGCGCACCAGGTAGCTCTACCCCGT
  	CTGGTGCTACCGGTTCCCCAGGTGCTTCT
  	CCTGGTACTAGCTCTACCGGTTCTCCAGG
  	TAGCTCTACCCCGTCTGGTGCTACTGGCT
  	CTCCA
  LCW462_r43	GGTTCTACTAGCTCTACTGCAGAATCTCC	521	GSTSSTAESPGPGTSPSG	522
  	GGGCCCAGGTACCTCTCCTAGCGGTGAA		ESSTAPGTSPSGESSTAP
  	TCTTCTACCGCTCCAGGTACTTCTCCGAG		GSTSSTAESPGPGSTSST
  	CGGTGAATCTTCTACCGCTCCAGGTTCTA		AESPGPGTSTPESGSASP
  	CTAGCTCTACCGCTGAATCTCCGGGTCCA		GTSPSGESSTAPGSTSST
  	GGTTCTACCAGCTCTACTGCAGAATCTCC		AESPGPGTSTPESGSASP
  	TGGCCCAGGTACTTCTACTCCGGAAAGC		GSTSSTAESPGPGSTSESP
  	GGTTCCGCTTCTCCAGGTACTTCTCCTAG		SGTAPGTSPSGESSTAP
  	CGGTGAATCTTCTACCGCTCCAGGTTCTA
  	CCAGCTCTACTGCTGAATCTCCTGGCCCA
  	GGTACTTCTACCCCGGAAAGCGGCTCCG
  	CTTCTCCAGGTTCTACCAGCTCTACCGCT
  	GAATCTCCTGGCCCAGGTTCTACTAGCG
  	AATCTCCGTCTGGCACCGCACCAGGTAC
  	TTCCCCTAGCGGTGAATCTTCTACTGCAC
  	CA
  LCW462_r45	GGTACCTCTACTCCGGAAAGCGGTTCCG	523	GTSTPESGSASPGSTSESP	524
  	CATCTCCAGGTTCTACCAGCGAATCCCCG		SGTAPGSTSSTAESPGPG
  	TCTGGCACCGCACCAGGTTCTACTAGCTC		TSTEPSEGSAPGTSTEPSE
  	TACTGCTGAATCTCCGGGCCCAGGTACCT		GSAPGTSESATPESGPGT
  	CTACTGAACCTTCCGAAGGCAGCGCTCC		SESATPESGPGTSTEPSE
  	AGGTACCTCTACCGAACCGTCCGAGGGC		GSAPGTSTEPSEGSAPGT
  	AGCGCACCAGGTACTTCTGAAAGCGCAA		SESATPESGPGTSTEPSE
  	CCCCTGAATCCGGTCCAGGTACCTCTGA		GSAPGTSTEPSEGSAP
  	AAGCGCTACTCCGGAGTCTGGCCCAGGT
  	ACCTCTACTGAACCGTCTGAGGGTAGCG
  	CTCCAGGTACTTCTACTGAACCGTCCGAA
  	GGTAGCGCACCAGGTACTTCTGAAAGCG
  	CTACTCCGGAGTCCGGTCCAGGTACCTCT
  	ACCGAACCGTCCGAAGGCAGCGCTCCAG
  	GTACTTCTACTGAACCTTCTGAGGGTAGC
  	GCTCCC
  LCW462_r47	GGTACCTCTACCGAACCGTCCGAGGGTA	525	GTSTEPSEGSAPGTSTEP	526
  	GCGCACCAGGTACCTCTACTGAACCGTC		SEGSAPGSEPATSGSETP
  	TGAGGGTAGCGCTCCAGGTAGCGAACCG		GTSTEPSEGSAPGTSESA
  	GCAACCTCCGGTTCTGAAACTCCAGGTA		TPESGPGTSESATPESGP
  	CTTCTACTGAACCGTCTGAAGGTAGCGC		GASPGTSSTGSPGSSPSA
  	ACCAGGTACTTCTGAAAGCGCAACCCCG		STGTGPGSSTPSGATGSP
  	GAATCCGGCCCAGGTACCTCTGAAAGCG		GSSTPSGATGSPGSSTPS
  	CAACCCCGGAGTCCGGCCCAGGTGCATC		GATGSPGASPGTSSTGSP
  	TCCGGGTACTAGCTCTACCGGTTCTCCAG
  	GTTCTAGCCCTTCTGCTTCCACTGGTACC
  	GGCCCAGGTAGCTCTACCCCGTCTGGTG
  	CTACTGGTTCCCCAGGTAGCTCTACTCCG
  	TCTGGTGCAACCGGTTCCCCAGGTAGCTC
  	TACTCCTTCTGGTGCTACTGGCTCCCCAG
  	GTGCATCCCCTGGCACCAGCTCTACCGGT
  	TCTCCA
  LCW462_r54	GGTAGCGAACCGGCAACCTCTGGCTCTG	527	GSEPATSGSETPGSEPAT	528
  	AAACTCCAGGTAGCGAACCTGCAACCTC		SGSETPGTSTEPSEGSAP
  	CGGCTCTGAAACCCCAGGTACTTCTACTG		GSEPATSGSETPGTSESA
  	AACCTTCTGAGGGCAGCGCACCAGGTAG		TPESGPGTSTEPSEGSAP
  	CGAACCTGCAACCTCTGGCTCTGAAACC		GSSTPSGATGSPGSSTPS
  	CCAGGTACCTCTGAAAGCGCTACTCCTG		GATGSPGASPGTSSTGSP
  	AATCTGGCCCAGGTACTTCTACTGAACC		GSSTPSGATGSPGASPGT
  	GTCCGAGGGCAGCGCACCAGGTAGCTCT		SSTGSPGSSTPSGATGSP
  	ACTCCGTCTGGTGCTACCGGCTCTCCAGG
  	TAGCTCTACCCCTTCTGGTGCAACCGGCT
  	CCCCAGGTGCTTCTCCGGGTACCAGCTCT
  	ACTGGTTCTCCAGGTAGCTCTACCCCGTC
  	TGGTGCTACCGGTTCCCCAGGTGCTTCTC
  	CTGGTACTAGCTCTACCGGTTCTCCAGGT
  	AGCTCTACCCCGTCTGGTGCTACTGGCTC
  	TCCA
  LCW462_r55	GGTACTTCTACCGAACCGTCCGAGGGCA	529	GTSTEPSEGSAPGTSTEP	530
  	GCGCTCCAGGTACTTCTACTGAACCTTCT		SEGSAPGTSTEPSEGSAP
  	GAAGGCAGCGCTCCAGGTACTTCTACTG		GTSESATPESGPGTSTEP
  	AACCTTCCGAAGGTAGCGCACCAGGTAC		SEGSAPGTSTEPSEGSAP
  	TTCTGAAAGCGCTACTCCGGAGTCCGGT		GSTSESPSGTAPGTSPSG
  	CCAGGTACCTCTACCGAACCGTCCGAAG		ESSTAPGTSPSGESSTAP
  	GCAGCGCTCCAGGTACTTCTACTGAACCT		GSPAGSPTSTEEGTSESA
  	TCTGAGGGTAGCGCTCCAGGTTCTACTA		TPESGPGTSTEPSEGSAP
  	GCGAATCTCCGTCTGGCACTGCTCCAGGT
  	ACTTCTCCTAGCGGTGAATCTTCTACCGC
  	TCCAGGTACTTCCCCTAGCGGCGAATCTT
  	CTACCGCTCCAGGTAGCCCGGCTGGCTCT
  	CCTACCTCTACTGAGGAAGGTACTTCTGA
  	AAGCGCTACTCCTGAGTCTGGTCCAGGT
  	ACCTCTACTGAACCGTCCGAAGGTAGCG
  	CTCCA
  LCW462_r57	GGTACTTCTACTGAACCTTCCGAAGGTA	531	GTSTEPSEGSAPGSEPAT	532
  	GCGCTCCAGGTAGCGAACCTGCTACTTCT		SGSETPGSPAGSPTSTEE
  	GGTTCTGAAACCCCAGGTAGCCCGGCTG		GSPAGSPTSTEEGTSESA
  	GCTCTCCGACCTCCACCGAGGAAGGTAG		TPESGPGTSTEPSEGSAP
  	CCCGGCAGGCTCTCCGACCTCTACTGAG		GTSTEPSEGSAPGTSTEP
  	GAAGGTACTTCTGAAAGCGCAACCCCGG		SEGSAPGTSESATPESGP
  	AGTCCGGCCCAGGTACCTCTACCGAACC		GSSTPSGATGSPGSSPSA
  	GTCTGAGGGCAGCGCACCAGGTACCTCT		STGTGPGASPGTSSTGSP
  	ACTGAACCTTCCGAAGGCAGCGCTCCAG
  	GTACCTCTACCGAACCGTCCGAGGGCAG
  	CGCACCAGGTACTTCTGAAAGCGCAACC
  	CCTGAATCCGGTCCAGGTAGCTCTACTCC
  	GTCTGGTGCAACCGGCTCCCCAGGTTCTA
  	GCCCGTCTGCTTCCACTGGTACTGGCCCA
  	GGTGCTTCCCCGGGCACCAGCTCTACTG
  	GTTCTCCA
  LCW462_r61	GGTAGCGAACCGGCTACTTCCGGCTCTG	533	GSEPATSGSETPGSPAGS	534
  	AGACTCCAGGTAGCCCTGCTGGCTCTCC		PTSTEEGTSESATPESGP
  	GACCTCTACCGAAGAAGGTACCTCTGAA		GTSTEPSEGSAPGTSTEP
  	AGCGCTACCCCTGAGTCTGGCCCAGGTA		SEGSAPGTSESATPESGP
  	CCTCTACTGAACCTTCCGAAGGCAGCGC		GTSTPESGSASPGSTSESP
  	TCCAGGTACCTCTACCGAACCGTCCGAG		SGTAPGSTSSTAESPGPG
  	GGCAGCGCACCAGGTACTTCTGAAAGCG		TSESATPESGPGTSTEPSE
  	CAACCCCTGAATCCGGTCCAGGTACCTCT		GSAPGTSTEPSEGSAP
  	ACTCCGGAAAGCGGTTCCGCATCTCCAG
  	GTTCTACCAGCGAATCCCCGTCTGGCACC
  	GCACCAGGTTCTACTAGCTCTACTGCTGA
  	ATCTCCGGGCCCAGGTACTTCTGAAAGC
  	GCTACTCCGGAGTCCGGTCCAGGTACCT
  	CTACCGAACCGTCCGAAGGCAGCGCTCC
  	AGGTACTTCTACTGAACCTTCTGAGGGTA
  	GCGCTCCA
  LCW462_r64	GGTACTTCTACCGAACCGTCCGAGGGCA	535	GTSTEPSEGSAPGTSTEP	536
  	GCGCTCCAGGTACTTCTACTGAACCTTCT		SEGSAPGTSTEPSEGSAP
  	GAAGGCAGCGCTCCAGGTACTTCTACTG		GTSTEPSEGSAPGTSESA
  	AACCTTCCGAAGGTAGCGCACCAGGTAC		TPESGPGTSESATPESGP
  	CTCTACCGAACCGTCTGAAGGTAGCGCA		GTPGSGTASSSPGSSTPS
  	CCAGGTACCTCTGAAAGCGCAACTCCTG		GATGSPGASPGTSSTGSP
  	AGTCCGGTCCAGGTACTTCTGAAAGCGC		GSTSSTAESPGPGTSPSG
  	AACCCCGGAGTCTGGCCCAGGTACTCCT		ESSTAPGTSTPESGSASP
  	GGCAGCGGTACCGCATCTTCCTCTCCAG
  	GTAGCTCTACTCCGTCTGGTGCAACTGGT
  	TCCCCAGGTGCTTCTCCGGGTACCAGCTC
  	TACCGGTTCTCCAGGTTCCACCAGCTCTA
  	CTGCTGAATCTCCTGGTCCAGGTACCTCT
  	CCTAGCGGTGAATCTTCTACTGCTCCAGG
  	TACTTCTACTCCTGAAAGCGGCTCTGCTT
  	CTCCA
  LCW462_r67	GGTAGCCCGGCAGGCTCTCCGACCTCTA	537	GSPAGSPTSTEEGTSESA	538
  	CTGAGGAAGGTACTTCTGAAAGCGCAAC		TPESGPGTSTEPSEGSAP
  	CCCGGAGTCCGGCCCAGGTACCTCTACC		GTSESATPESGPGSEPAT
  	GAACCGTCTGAGGGCAGCGCACCAGGTA		SGSETPGTSTEPSEGSAP
  	CTTCTGAAAGCGCAACCCCTGAATCCGG		GSPAGSPTSTEEGTSTEP
  	TCCAGGTAGCGAACCGGCTACTTCTGGC		SEGSAPGTSTEPSEGSAP
  	TCTGAGACTCCAGGTACTTCTACCGAACC		GTSTEPSEGSAPGTSTEP
  	GTCCGAAGGTAGCGCACCAGGTAGCCCG		SEGSAPGTSTEPSEGSAP
  	GCTGGTTCTCCGACTTCCACCGAGGAAG
  	GTACCTCTACTGAACCTTCTGAGGGTAGC
  	GCTCCAGGTACCTCTACTGAACCTTCCGA
  	AGGCAGCGCTCCAGGTACTTCTACCGAA
  	CCGTCCGAGGGCAGCGCTCCAGGTACTT
  	CTACTGAACCTTCTGAAGGCAGCGCTCC
  	AGGTACTTCTACTGAACCTTCCGAAGGT
  	AGCGCACCA
  LCW462_r69	GGTACTTCTCCGAGCGGTGAATCTTCTAC	539	GTSPSGESSTAPGSTSST	540
  	CGCACCAGGTTCTACTAGCTCTACCGCTG		AESPGPGTSPSGESSTAP
  	AATCTCCGGGCCCAGGTACTTCTCCGAG		GTSESATPESGPGTSTEP
  	CGGTGAATCTTCTACTGCTCCAGGTACCT		SEGSAPGTSTEPSEGSAP
  	CTGAAAGCGCTACTCCGGAGTCTGGCCC		GSSPSASTGTGPGSSTPS
  	AGGTACCTCTACTGAACCGTCTGAGGGT		GATGSPGASPGTSSTGSP
  	AGCGCTCCAGGTACTTCTACTGAACCGTC		GTSTPESGSASPGTSPSG
  	CGAAGGTAGCGCACCAGGTTCTAGCCCT		ESSTAPGTSPSGESSTAP
  	TCTGCATCTACTGGTACTGGCCCAGGTAG
  	CTCTACTCCTTCTGGTGCTACCGGCTCTC
  	CAGGTGCTTCTCCGGGTACTAGCTCTACC
  	GGTTCTCCAGGTACTTCTACTCCGGAAAG
  	CGGTTCCGCATCTCCAGGTACTTCTCCTA
  	GCGGTGAATCTTCTACTGCTCCAGGTACC
  	TCTCCTAGCGGCGAATCTTCTACTGCTCCA
  LCW462_r70	GGTACCTCTGAAAGCGCTACTCCGGAGT	541	GTSESATPESGPGTSTEP	542
  	CTGGCCCAGGTACCTCTACTGAACCGTCT		SEGSAPGTSTEPSEGSAP
  	GAGGGTAGCGCTCCAGGTACTTCTACTG		GSPAGSPTSTEEGSPAGS
  	AACCGTCCGAAGGTAGCGCACCAGGTAG		PTSTEEGTSTEPSEGSAP
  	CCCTGCTGGCTCTCCGACTTCTACTGAGG		GSSPSASTGTGPGSSTPS
  	AAGGTAGCCCGGCTGGTTCTCCGACTTCT		GATGSPGSSTPSGATGSP
  	ACTGAGGAAGGTACTTCTACCGAACCTT		GSEPATSGSETPGTSESA
  	CCGAAGGTAGCGCTCCAGGTTCTAGCCC		TPESGPGSEPATSGSETP
  	TTCTGCTTCCACCGGTACTGGCCCAGGTA
  	GCTCTACCCCTTCTGGTGCTACCGGCTCC
  	CCAGGTAGCTCTACTCCTTCTGGTGCAAC
  	TGGCTCTCCAGGTAGCGAACCGGCAACT
  	TCCGGCTCTGAAACCCCAGGTACTTCTGA
  	AAGCGCTACTCCTGAGTCTGGCCCAGGT
  	AGCGAACCTGCTACCTCTGGCTCTGAAA
  	CCCCA
  LCW462_r72	GGTACTTCTACCGAACCGTCCGAAGGCA	543	GTSTEPSEGSAPGTSTEP	544
  	GCGCTCCAGGTACCTCTACTGAACCTTCC		SEGSAPGTSTEPSEGSAP
  	GAGGGCAGCGCTCCAGGTACCTCTACCG		GSSTPSGATGSPGASPGT
  	AACCTTCTGAAGGTAGCGCACCAGGTAG		SSTGSPGSSTPSGATGSP
  	CTCTACCCCGTCTGGTGCTACCGGTTCCC		GTSESATPESGPGSEPAT
  	CAGGTGCTTCTCCTGGTACTAGCTCTACC		SGSETPGTSTEPSEGSAP
  	GGTTCTCCAGGTAGCTCTACCCCGTCTGG		GSTSESPSGTAPGSTSESP
  	TGCTACTGGCTCTCCAGGTACTTCTGAAA		SGTAPGTSTPESGSASP
  	GCGCAACCCCTGAATCCGGTCCAGGTAG
  	CGAACCGGCTACTTCTGGCTCTGAGACTC
  	CAGGTACTTCTACCGAACCGTCCGAAGG
  	TAGCGCACCAGGTTCTACTAGCGAATCT
  	CCTTCTGGCACTGCACCAGGTTCTACCAG
  	CGAATCTCCGTCTGGCACTGCACCAGGT
  	ACCTCTACCCCTGAAAGCGGTTCCGCTTC
  	TCCA
  LCW462_r73	GGTACCTCTACTCCTGAAAGCGGTTCTGC	545	GTSTPESGSASPGSTSST	546
  	ATCTCCAGGTTCCACTAGCTCTACCGCAG		AESPGPGSTSSTAESPGP
  	AATCTCCGGGCCCAGGTTCTACTAGCTCT		GSSPSASTGTGPGSSTPS
  	ACTGCTGAATCTCCTGGCCCAGGTTCTAG		GATGSPGASPGTSSTGSP
  	CCCTTCTGCATCTACTGGTACTGGCCCAG		GSEPATSGSETPGTSESA
  	GTAGCTCTACTCCTTCTGGTGCTACCGGC		TPESGPGSPAGSPTSTEE
  	TCTCCAGGTGCTTCTCCGGGTACTAGCTC		GSTSESPSGTAPGSTSESP
  	TACCGGTTCTCCAGGTAGCGAACCGGCA		SGTAPGTSTPESGSASP
  	ACCTCCGGCTCTGAAACCCCAGGTACCT
  	CTGAAAGCGCTACTCCTGAATCCGGCCC
  	AGGTAGCCCGGCAGGTTCTCCGACTTCC
  	ACTGAGGAAGGTTCTACTAGCGAATCTC
  	CTTCTGGCACTGCACCAGGTTCTACCAGC
  	GAATCTCCGTCTGGCACTGCACCAGGTA
  	CCTCTACCCCTGAAAGCGGTTCCGCTTCT
  	CCC
  LCW462_r78	GGTAGCCCGGCTGGCTCTCCTACCTCTAC	547	GSPAGSPTSTEEGTSESA	548
  	TGAGGAAGGTACTTCTGAAAGCGCTACT		TPESGPGTSTEPSEGSAP
  	CCTGAGTCTGGTCCAGGTACCTCTACTGA		GSTSESPSGTAPGSTSESP
  	ACCGTCCGAAGGTAGCGCTCCAGGTTCT		SGTAPGTSPSGESSTAPG
  	ACCAGCGAATCTCCTTCTGGCACCGCTCC		TSTEPSEGSAPGSPAGSP
  	AGGTTCTACTAGCGAATCCCCGTCTGGTA		TSTEEGTSTEPSEGSAPG
  	CCGCACCAGGTACTTCTCCTAGCGGCGA		SEPATSGSETPGTSESAT
  	ATCTTCTACCGCACCAGGTACCTCTACCG		PESGPGTSTEPSEGSAP
  	AACCTTCCGAAGGTAGCGCTCCAGGTAG
  	CCCGGCAGGTTCTCCTACTTCCACTGAGG
  	AAGGTACTTCTACCGAACCTTCTGAGGG
  	TAGCGCACCAGGTAGCGAACCTGCAACC
  	TCTGGCTCTGAAACCCCAGGTACCTCTGA
  	AAGCGCTACTCCTGAATCTGGCCCAGGT
  	ACTTCTACTGAACCGTCCGAGGGCAGCG
  	CACCA
  LCW462_r79	GGTACCTCTACCGAACCTTCCGAAGGTA	549	GTSTEPSEGSAPGSPAGS	550
  	GCGCTCCAGGTAGCCCGGCAGGTTCTCC		PTSTEEGTSTEPSEGSAP
  	TACTTCCACTGAGGAAGGTACTTCTACCG		GTSPSGESSTAPGTSPSG
  	AACCTTCTGAGGGTAGCGCACCAGGTAC		ESSTAPGTSPSGESSTAP
  	CTCCCCTAGCGGCGAATCTTCTACTGCTC		GSTSESPSGTAPGSTSESP
  	CAGGTACCTCTCCTAGCGGCGAATCTTCT		SGTAPGTSTPESGSASPG
  	ACCGCTCCAGGTACCTCCCCTAGCGGTG		SEPATSGSETPGTSESAT
  	AATCTTCTACCGCACCAGGTTCTACCAGC		PESGPGTSTEPSEGSAP
  	GAATCCCCTTCTGGTACTGCTCCAGGTTC
  	TACCAGCGAATCCCCTTCTGGCACCGCA
  	CCAGGTACTTCTACCCCTGAAAGCGGCT
  	CCGCTTCTCCAGGTAGCGAACCTGCAAC
  	CTCTGGCTCTGAAACCCCAGGTACCTCTG
  	AAAGCGCTACTCCTGAATCTGGCCCAGG
  	TACTTCTACTGAACCGTCCGAGGGCAGC
  	GCACCA
  LCW462_r87	GGTAGCGAACCGGCAACCTCTGGCTCTG	551	GSEPATSGSETPGTSESA	552
  	AAACCCCAGGTACCTCTGAAAGCGCTAC		TPESGPGTSESATPESGP
  	TCCGGAATCTGGTCCAGGTACTTCTGAA		GTSPSGESSTAPGSTSST
  	AGCGCTACTCCGGAATCCGGTCCAGGTA		AESPGPGTSPSGESSTAP
  	CTTCTCCGAGCGGTGAATCTTCTACCGCA		GSTSESPSGTAPGTSPSG
  	CCAGGTTCTACTAGCTCTACCGCTGAATC		ESSTAPGSTSSTAESPGP
  	TCCGGGCCCAGGTACTTCTCCGAGCGGT		GSSTPSGATGSPGSSTPS
  	GAATCTTCTACTGCTCCAGGTTCTACTAG		GATGSPGSSTPSGANWLS
  	CGAATCCCCGTCTGGTACTGCTCCAGGTA
  	CTTCCCCTAGCGGTGAATCTTCTACTGCT
  	CCAGGTTCTACCAGCTCTACCGCAGAAT
  	CTCCGGGTCCAGGTAGCTCTACTCCGTCT
  	GGTGCAACCGGTTCCCCAGGTAGCTCTA
  	CCCCTTCTGGTGCAACCGGCTCCCCAGGT
  	AGCTCTACCCCTTCTGGTGCAAACTGGCT
  	CTCC
  LCW462_r88	GGTAGCCCTGCTGGCTCTCCGACTTCTAC	553	GSPAGSPTSTEEGSPAGS	554
  	TGAGGAAGGTAGCCCGGCTGGTTCTCCG		PTSTEEGTSTEPSEGSAP
  	ACTTCTACTGAGGAAGGTACTTCTACCG		GTSTEPSEGSAPGTSTEP
  	AACCTTCCGAAGGTAGCGCTCCAGGTAC		SEGSAPGTSESATPESGP
  	CTCTACTGAACCTTCCGAAGGCAGCGCT		GASPGTSSTGSPGSSTPS
  	CCAGGTACCTCTACCGAACCGTCCGAGG		GATGSPGASPGTSSTGSP
  	GCAGCGCACCAGGTACTTCTGAAAGCGC		GSSTPSGATGSPGTPGSG
  	AACCCCTGAATCCGGTCCAGGTGCATCT		TASSSPGSSTPSGATGSP
  	CCTGGTACCAGCTCTACCGGTTCTCCAGG
  	TAGCTCTACTCCTTCTGGTGCTACTGGCT
  	CTCCAGGTGCTTCCCCGGGTACCAGCTCT
  	ACCGGTTCTCCAGGTAGCTCTACCCCGTC
  	TGGTGCTACTGGTTCTCCAGGTACTCCGG
  	GCAGCGGTACTGCTTCTTCCTCTCCAGGT
  	AGCTCTACCCCTTCTGGTGCTACTGGCTC
  	TCCA
  LCW462_r89	GGTAGCTCTACCCCGTCTGGTGCTACTGG	555	GSSTPSGATGSPGTPGSG	556
  	TTCTCCAGGTACTCCGGGCAGCGGTACT		TASSSPGSSTPSGATGSP
  	GCTTCTTCCTCTCCAGGTAGCTCTACCCC		GSPAGSPTSTEEGTSESA
  	TTCTGGTGCTACTGGCTCTCCAGGTAGCC		TPESGPGTSTEPSEGSAP
  	CGGCTGGCTCTCCTACCTCTACTGAGGAA		GTSESATPESGPGSEPAT
  	GGTACTTCTGAAAGCGCTACTCCTGAGTC		SGSETPGTSESATPESGP
  	TGGTCCAGGTACCTCTACTGAACCGTCCG		GTSTEPSEGSAPGTSESA
  	AAGGTAGCGCTCCAGGTACCTCTGAAAG		TPESGPGTSESATPESGP
  	CGCAACTCCTGAGTCTGGCCCAGGTAGC
  	GAACCTGCTACCTCCGGCTCTGAGACTCC
  	AGGTACCTCTGAAAGCGCAACCCCGGAA
  	TCTGGTCCAGGTACTTCTACTGAACCGTC
  	TGAAGGTAGCGCACCAGGTACTTCTGAA
  	AGCGCAACCCCGGAATCCGGCCCAGGTA
  	CCTCTGAAAGCGCAACCCCGGAGTCCGG
  	CCCA

Example 7Construction of XTEN_AM288
• The entire library LCW0462 was dimerized as described in Example 6 resulting in a library of XTEN_AM288 clones designated LCW0463. 1512 isolates from library LCW0463 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 40 preferred XTEN_AM288 segments were chosen for the construction of multifunctional proteins that contain multiple XTEN segments with 288 amino acid residues.
Example 8Construction of XTEN_AM432
• We generated a library of XTEN_AM432 segments by recombining segments from library LCW0462 of XTEN_AM144 segments and segments from library LCW0463 of XTEN_AM288 segments. This new library of XTEN_AM432 segment was designated LCW0464. Plasmid was isolated from cultures ofE. coliharboring LCW0462 and LCW0463, respectively. 1512 isolates from library LCW0464 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 39 preferred XTEN_AM432 segment were chosen for the construction of longer XTENs and for the construction of multifunctional proteins that contain multiple XTEN segments with 432 amino acid residues.
• In parallel we constructed library LMS0100 of XTEN_AM432 segments using preferred segments of XTEN_AM144 and XTENAM288. Screening of this library yielded 4 isolates that were selected for further construction
Example 9Construction of XTEN_AM875
• The stuffer vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification.
• We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnIfor P: AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 557) and the non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev: CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 558) for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG (SEQ ID NO: 559) and has the restriction enzyme AscI recognition nucleotide sequence GGCGCGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCW0359 prepared above to yield pCW0466 containing SI-A. We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-A segments from pCW0466 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0479.
• We generated a library of XTEN_AM875 segments by recombining segments from library LCW0479 of XTEN_AM443 segments and 43 preferred XTEN_AM432 segments from Example 8 using the same dimerization process described in Example 5. This new library of XTEN_AM875 segment was designated LCW0481.
Example 10Construction of XTEN_AM1318
• We annealed the phosphorylated oligonucleotide BsaI-FseI-KpnIforP: AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 560) and the non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev: CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 561) for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG (SEQ ID NO: 562) and has the restriction enzyme FseI recognition nucleotide sequence GGCCGGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCW0359 as used in Example 9 to yield pCW0467 containing SI-B. We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-B segments from pCW0467 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0480.
• We generated a library of XTEN_AM1318 segments by recombining segments from library LCW0480 of XTEN_AM443 segments and segments from library LCW0481 of XTEN_AM875 segments using the same dimerization process as in Example 5. This new library of XTEN_AM1318 segment was designated LCW0487.
Example 11Construction of XTEN_AD864
• Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AD864 sequences starting from segments of XTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AD864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_AD576 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20 h was measured.
Example 12Construction of XTEN_AF864
• Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AF864 sequences starting from segments of XTEN_AF36 listed in Example 3. These sequences were assembled as described in Example 5. Several isolates from XTEN_AF864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_AF540 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20 h was measured. A full length clone of XTEN_AF864 had excellent solubility and showed half-life exceeding 60 h in cynomolgus monkeys. A second set of XTEN_AF sequences was assembled including a sequencing island as described in Example 9.
Example 13Construction of XTEN_AG864
• Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AG864 sequences starting from segments of XTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AG864 were evaluated and found to show good expression and excellent solubility under physiological conditions. A full-length clone of XTEN_AG864 had excellent solubility and showed half-life exceeding 60 h in cynomolgus monkeys.
Example 14Construction of N-Terminal Extensions of XTEN-Construction and Screening of 12Mer Addition Libraries
• This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of fusion proteins without the presence of a helper domain. Historically expression of proteins with XTEN at the N-terminus was poor, yielding values that would essentially undetectable in the GFP fluorescence assay (<25% of the expression with the N-terminal CBD helper domain). To create diversity at the codon level, seven amino acid sequences were selected and prepared with a diversity of codons. Seven pairs of oligonucleotides encoding 12 amino acids with codon diversities were designed, annealed and ligated into the NdeI/BsaI restriction enzyme digested stiffer vector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of seven libraries. The resulting clones have N-terminal XTEN 12mers fused in-frame to XTEN_AM875-GFP to allow use of GFP fluorescence for screening the expression. Individual colonies from the seven created libraries were picked and grown overnight to saturation in 500 μl of super broth media in a 96 deep well plate. The number of colonies picked ranged from approximately half to a third of the theoretical diversity of the library (see Table 14).

• TABLE 14
  Theoretical Diversity and
  Sampling Numbers for
  12mer Addition Libraries. The amino
  acid residues with randomized
  codons are underlined.

  				Theo-
  			SEQ	retical
  	Motif	Amino Acid	ID	Diver-	Number
  Library	Family	Sequence	NO:	sity	screened

  LCW546	AE12	MASPAGSPTSTEE	563	572	2 plates
  					(168)
  LCW547	AE12	MATSESATPESGP	564	1536	5 plates
  					(420)
  LCW548	AF12	MATSPSGESSTAP	565	192	2 plates
  					(168)
  LCW549	AF12	MESTSSTAESPGP	566	384	2 plates
  					(168)
  LCW552	AG12	MASSTPSGATGSP	567	384	2 plates
  					(168)
  LCW553	AG12	MEASPGTSSTGSP	568	384	2 plates
  					(168)
  LCW554	(CBD-	MASTPESGSSG	569	32	1 plate
  	like)				(84)

• The saturated overnight cultures were used to inoculate fresh 500 μl cultures in auto-induction media in which they were grown overnight at 26° C. These expression cultures were then assayed using a fluorescence plate reader (excitation 395 nm, emission 510 nm) to determine the amount of GFP reporter present (seeFIG. 28 for results of expression assays). The results indicated that while median expression levels were approximately half of the expression levels compared to the “benchmark” CBD N-terminal helper domain, the best clones from the libraries were much closer to the benchmarks, indicating that further optimization around those sequences was warranted. This is in contrast to previous XTEN versions that were <25% of the expression levels of the CBD N-terminal benchmark. The results also show that the libraries starting with amino acids MA had better expression levels than those beginning with ME. This was most apparent when looking at the best clones, which were closer to the benchmarks as they mostly start with MA. Of the 176 clones within 33% of the CBD-AM875 benchmark, 87% begin with MA, where as only 75% of the sequences in the libraries beginning with MA, a clear over representation of the clones beginning with MA at the highest level of expression. 96 of the best clones were sequenced to confirm identity and twelve sequences (see Table 15), 4 from LCW546, 4 from LCW547 and 4 from LCW552 were selected for further optimization.

• TABLE 15
  Advanced 12mer DNA Nucleotide Sequences

  		SEQ
  		ID
  Clone	DNA Nucleotide Sequence	NO:
  LCW546_02	ATGGCTAGTCCGGCTGGCTCTCCGACCTCCACTGAGGAAGGTACTTCTACT	570
  LCW546_06	ATGGCTAGTCCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACTTCTACT	571
  LCW546_07	ATGGCTAGTCCAGCAGGCTCTCCTACCTCCACCGAGGAAGGTACTTCTACT	572
  LCW546_09	ATGGCTAGTCCTGCTGGCTCTCCGACCTCTACTGAGGAAGGTACTTCTACT	573
  LCW547_03	ATGGCTACATCCGAAAGCGCAACCCCTGAGTCCGGTCCAGGTACTTCTACT	574
  LCW547_06	ATGGCTACATCCGAAAGCGCAACCCCTGAATCTGGTCCAGGTACTTCTACT	575
  LCW547_10	ATGGCTACGTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTACT	576
  LCW547_17	ATGGCTACGTCCGAAAGCGCTACCCCTGAATCCGGTCCAGGTACTTCTACT	577
  LCW552_03	ATGGCTAGTTCTACCCCGTCTGGTGCAACCGGTTCCCCAGGTACTTCTACT	578
  LCW552_05	ATGGCTAGCTCCACTCCGTCTGGTGCTACCGGTTCCCCAGGTACTTCTACT	579
  LCW552_10	ATGGCTAGCTCTACTCCGTCTGGTGCTACTGGTTCCCCAGGTACTTCTACT	580
  LCW552_11	ATGGCTAGTTCTACCCCTTCTGGTGCTACTGGTTCTCCAGGTACTTCTACT	581

Example 15Construction of N-Terminal Extensions of XTEN-Construction and Screening ofLibraries Optimizing Codons 3 and 4
• This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain. With preferences for the first two codons established (see Example supra), the third and fourth codons were randomized to determine preferences. Three libraries, based upon best clones from LCW546, LCW547 and LCW552, were designed with the third and fourth residues modified such that all combinations of allowable XTEN codons were present at these positions (seeFIG. 29). In order to include all the allowable XTEN codons for each library, nine pairs of oligonucleotides encoding 12 amino acids with codon diversities of third and fourth residues were designed, annealed and ligated into the NdeI/BsaI restriction enzyme digested stuffer vector pCW0551 (Stuffer-XTEN_AM875-GFP), and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of three libraries LCW0569-571. With 24 XTEN codons the theoretical diversity of each library is 576 unique clones. A total of 504 individual colonies from the three created libraries were picked and grown overnight to saturation in 500 μl of super broth media in a 96 deep well plate. This provided sufficient coverage to understand relative library performance and sequence preferences. The saturated overnight cultures were used to inoculate new 500 μl cultures in auto-induction media in which were grown overnight at 26° C. These expression cultures were then assayed using a fluorescence plate reader (excitation 395 nm, emission 510 nm) to determine the amount of GFP reporter present. The top 75 clones from the screen were sequenced and retested for GFP reporter expression versus the benchmark samples (seeFIG. 28). 52 clones yielded usable sequencing data and were used for subsequent analysis. The results were broken down by library and indicate that LCW546 was the superior library. The results are presented in Table 16. Surprisingly, it was discovered that base-lined fluorescence readings for the best clones were ˜900 AU, whereas the CBD N-terminal benchmark was only ˜600 AU. This indicates that this library had instituted an approximately 33% improvement over the best clones from the previous library which were approximately equal in expression to the CBD N-terminal benchmark (Example 14).

• TABLE 16
  Third and Fourth Codon Optimization Library Comparison

  		LCW569	LCW570	LCW571

  	N	21	15	16
  	Mean	628	491	537
  	Fluorescence (AU)
  	SD	173	71	232
  	CV	28%	15%	43%

• Further trends were seen in the data showing preferences for particular codons at the third and fourth position. Within the LCW569 library the glutamate codon GAA at the third position and the threonine codon ACT were associated with higher expression as seen in Table 17.

• TABLE 17
  Preferred Third and Fourth Codons inLCW569

  	3 =GAA	Rest		4 =ACT	Rest

  N

  	8	13	4	17
  	Mean	749	554	744	601
  	Fluorescence (AU)
  	SD	234	47	197	162
  	CV	31%	9%	26%	27%

• Additionally, the retest of the top 75 clones indicated that several were now superior to the benchmark clones.
Example 16Construction of N-Terminal Extensions of XTEN-Construction and Screening of Combinatorial 12Mer and 36Mer Libraries
• This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain. With preferences for the first two codons established (see Example supra), the N-terminus was examined in a broader context by combining the 12 selected 12mer sequences (see Example supra) at the very N-terminus followed by 125 previously constructed 36mer segments (see example supra) in a combinatorial manner. This created novel 48mers at the N-terminus of the XTEN protein and enabled the assessment of the impact of longer-range interactions at the N-terminus on expression of the longer sequences (FIG. 29). Similar to the dimerization procedures used to assemble 36mers (see Example infra), the plasmids containing the 125 selected 36mer segments were digested with restriction enzymes BbsI/NcoI and the appropriate fragment was gel-purified. The plasmid from clone AC94 (CBD-XTEN_AM875-GFP) was also digested with BsaI/NcoI and the appropriate fragments were gel-purified. These fragments were ligated together and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of the library LCW0579, which also served as the vector forfurther cloning 12 selected 12mers at the very N-terminus. The plasmids of LCW0579 were digested with NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. 12 pairs of oligonucleotides encoding 12 selected 12mer sequences were designed, annealed and ligated with the NdeI/EcoRI/BsaI digested LCW0579 vector, and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of the library LCW0580. With a theoretical diversity of 1500 unique clones, a total of 1512 individual colonies from the created library were picked and grown overnight to saturation in 500 μl of super broth media in a 96 deep well plate. This provided sufficient coverage to understand relative library performance and sequence preferences. The saturated overnight cultures were used to inoculate new 500 μl cultures in auto-induction media that were grown overnight at 26° C. These expression cultures were then assayed using a fluorescence plate reader (excitation 395 nm, emission 510 nm) to determine the amount of GFP reporter present. The top 90 clones were sequenced and retested for GFP reporter expression. 83 clones yielded usable sequencing data and were used for subsequent analysis. The sequencing data was used to determine the lead 12mer that was present in each clone and the impact of each 12mer on expression was assessed. Clones LCW546_—06 and LCW546_—09 stood out as being the superior N-terminus (see Table 18).

• TABLE 18
  Relative Performance of Clones Starting with LCW546 06
  and LCW459 09

  		All		All
  	LCW546_06	Others	LCW546_09	Others

  N

  	11	72	9	74
  Mean	1100	752	988	775
  Fluorescence (AU)
  SD	275	154	179	202
  CV	25%	20%	18%	26%

• The sequencing and retest also revealed several instances of independent replicates of the same sequence in the data producing similar results, thus increasing confidence in the assay. Additionally, 10 clones with 6 unique sequences were superior to the benchmark clone. They are presented in Table 19. It was noted that these were the only occurrences of these sequences and in no case did one of these sequences occur and fail to beat the bench-mark clone. These six sequences were advanced for further optimization.

• TABLE 19
  Combinatorial 12mer and 36mer Clones Superior to Benchmark Clone

  		SEQ
  		ID
  Clone Name	First 60 codons	NO:	12mer Name	36mer Name
  LCW580_51	ATGGCTAGTCCTGCTGGCTCTCCAACCTC	582	LCW546_06	LCW0404_040
  	CACTGAGGAAGGTGCATCCCCGGGCACC
  	AGCTCTACCGGTTCTCCAGGTAGCTCTAC
  	CCCGTCTGGTGCTACCGGCTCTCCAGGTA
  	GCTCTACCCCGTCTGGTGCTACTGGCTCTC
  	CAGGTACTTCTACTGAACCGTCTGAAGGC
  	AGCGCA
  LCW580_81	ATGGCTAGTCCTGCTGGCTCTCCAACCTC	583	LCW546_06	LCW0404_040
  	CACTGAGGAAGGTGCATCCCCGGGCACC
  	AGCTCTACCGGTTCTCCAGGTAGCTCTAC
  	CCCGTCTGGTGCTACCGGCTCTCCAGGTA
  	GCTCTACCCCGTCTGGTGCTACTGGCTCTC
  	CAGGTACTTCTACTGAACCGTCTGAAGGC
  	AGCGCA
  LCW580_38	ATGGCTAGTCCTGCTGGCTCTCCAACCTC	584	LCW546_06	LCW0402_041
  	CACTGAGGAAGGTACTTCTACCGAACCGT
  	CCGAGGGTAGCGCACCAGGTAGCCCAGC
  	AGGTTCTCCTACCTCCACCGAGGAAGGTA
  	CTTCTACCGAACCGTCCGAGGGTAGCGCA
  	CCAGGTACTTCTACTGAACCGTCTGAAGG
  	CAGCGCA
  LCW580_63	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	585	LCW546_09	LCW0402_020
  	TACTGAGGAAGGTACTTCTACTGAACCGT
  	CTGAAGGCAGCGCACCAGGTAGCGAACC
  	GGCTACTTCCGGTTCTGAAACCCCAGGTA
  	GCCCAGCAGGTTCTCCAACTTCTACTGAA
  	GAAGGTACTTCTACTGAACCGTCTGAAGG
  	CAGCGCA
  LCW580_06	ATGGCTAGTCCTGCTGGCTCTCCAACCTC	586	LCW546_06	LCW0404_031
  	CACTGAGGAAGGTACCCCGGGTAGCGGT
  	ACTGCTTCTTCCTCTCCAGGTAGCTCTACC
  	CCTTCTGGTGCAACCGGCTCTCCAGGTGC
  	TTCTCCGGGCACCAGCTCTACCGGTTCTC
  	CAGGTACTTCTACTGAACCGTCTGAAGGC
  	AGCGCA
  LCW580_35	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	587	LCW546_09	LCW0402_020
  	TACTGAGGAAGGTACTTCTACTGAACCGT
  	CTGAAGGCAGCGCACCAGGTAGCGAACC
  	GGCTACTTCCGGTTCTGAAACCCCAGGTA
  	GCCCAGCAGGTTCTCCAACTTCTACTGAA
  	GAAGGTACTTCTACTGAACCGTCTGAAGG
  	CAGCGCA
  LCW580_67	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	588	LCW546_09	LCW0403_064
  	TACTGAGGAAGGTACCTCCCCTAGCGGCG
  	AATCTTCTACTGCTCCAGGTACCTCTCCTA
  	GCGGCGAATCTTCTACCGCTCCAGGTACC
  	TCCCCTAGCGGTGAATCTTCTACCGCACC
  	AGGTACTTCTACTGAACCGTCTGAAGGCA
  	GCGCA
  LCW580_13	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	589	LCW546_09	LCW0403_060
  	TACTGAGGAAGGTACCTCTACTCCGGAAA
  	GCGGTTCCGCATCTCCAGGTTCTACCAGC
  	GAATCCCCGTCTGGCACCGCACCAGGTTC
  	TACTAGCTCTACTGCTGAATCTCCGGGCC
  	CAGGTACTTCTACTGAACCGTCTGAAGGC
  	AGCGCA
  LCW580_88	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	590	LCW546_09	LCW0403_064
  	TACTGAGGAAGGTACCTCCCCTAGCGGCG
  	AATCTTCTACTGCTCCAGGTACCTCTCCTA
  	GCGGCGAATCTTCTACCGCTCCAGGTACC
  	TCCCCTAGCGGTGAATCTTCTACCGCACC
  	AGGTACTTCTACTGAACCGTCTGAAGGCA
  	GCGCA
  LCW580_11	ATGGCTAGTCCTGCTGGCTCTCCGACCTC	591	LCW546_09	LCW0403_060
  	TACTGAGGAAGGTACCTCTACTCCGGAAA
  	GCGGTTCCGCATCTCCAGGTTCTACCAGC
  	GAATCCCCGTCTGGCACCGCACCAGGTTC
  	TACTAGCTCTACTGCTGAATCTCCGGGCC
  	CAGGTACTTCTACTGAACCGTCTGAAGGC
  	AGCGCA

Example 17Construction of N-Terminal Extensions of XTEN-Construction and Screening of Combinatorial 12Mer and 36Mer Libraries for XTEN-AM875 and XTEN-AE864
• This example details a step in the optimization of the N-terminus of the XTEN protein to promote the initiation of translation to allow for expression of XTEN fusions at the N-terminus of proteins without the presence of a helper domain. With preferences for the first four codons (see Examples supra, and for the best pairing of N-terminal 12mers and 36mers (see Example supra) established, a combinatorial approach was undertaken to examine the union of these preferences. This created novel 48mers at the N-terminus of the XTEN protein and enabled the testing of the confluence of previous conclusions. Additionally, the ability of these leader sequences to be a universal solution for all XTEN proteins was assessed by placing the new 48mers in front of both XTEN-AE864 and XTEN-AM875. Instead of using all 125 clones of 36mer segment, the plasmids from 6 selected clones of 36mer segment with best GFP expression in the combinatorial library were digested with NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. The plasmids from clones AC94 (CBD-XTEN_AM875-GFP) and AC104 (CBD-XTEN_AE864-GFP) were digested with digested with NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. These fragments were ligated together and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of the libraries LCW0585 (—XTEN_AM875-GFP) and LCW0586 (—XTEN_AE864-GFP), which could also serve as the vectors for further cloning 8 selected 12mers at the very N-terminus The plasmids of LCW0585 and LCW0586 were digested with NdeI/EcoRI/BsaI and the appropriate fragments were gel-purified. 8 pairs of oligonucleotides encoding 8 selected 12mer sequences with best GFP expression in the previous (Generation 2) screening were designed, annealed and ligated with the NdeI/EcoRI/BsaI digested LCW0585 and LCW0586 vectors, and transformed intoE. coliBL21Gold(DE3) competent cells to obtain colonies of the final libraries LCW0587 (XTEN_AM923-GFP) and LCW0588 (XTEN_AE912-GFP). With a theoretical diversity of 48 unique clones, a total of 252 individual colonies from the created libraries were picked and grown overnight to saturation in 500 μl of super broth media in a 96 deep well plate. This provided sufficient coverage to understand relative library performance and sequence preferences. The saturated overnight cultures were used to inoculate new 500 μl cultures in auto-induction media in which were grown overnight at 26° C. These expression cultures were then assayed using a fluorescence plate reader (excitation 395 nm, emission 510 nm) to determine the amount of GFP reporter present. The top 36 clones were sequenced and retested for GFP reporter expression. 36 clones yielded usable sequencing data and these 36 were used for the subsequent analysis. The sequencing data determined the 12mer, the third codon, the fourth codon and the 36mer present in the clone and revealed that many of the clones were independent replicates of the same sequence. Additionally, the retest results for these clones are close in value, indicating the screening process was robust. Preferences for certain combinations at the N-terminus were seen and were consistently yielding higher fluorescence values approximately 50% greater than the benchmark controls (see Tables 20 and 21). These date support the conclusion that the inclusion of the sequences encoding the optimized N-terminal XTEN into the fusion protein genes conferred a marked enhancement on the expression of the fusion proteins.

• TABLE 20
  Preferred N-terminal Combinations for XTEN-AM875

  	Number of
  Clone Name	Replicates	12mer	36mer	Mean	SD	CV
  CBD-AM875	NA	NA	NA	1715	418	16%
  LCW587_08	7	LCW546_06_3 = GAA	LCW404_40	2333	572	18%
  LCW587_17	5	LCW546_09_3 = GAA	LCW403_64	2172	293	10%

• TABLE 21
  Preferred N-terminal Combinations for XTEN-AE864

  	Number of
  Clone Name	Replicates	12mer	36mer	Mean	SD	CV

  AC82	NA	NA	NA	1979	679	24%
  LCW588_14	8	LCW546_06_opt3	LCW404_31	2801	240	6%
  LCW588_27	2	LCW546_06_opt34	LCW404_40	2839	556	15%

• Notably, the preferred combination of the N-terminal for the XTEN-AM875 and the preferred combination for the XTEN-AE864 are not the same, indicating more complex interactions further than 150 bases from the initiation site influence expression levels. The sequences for the preferred nucleotide sequences are listed in Table 22 and the preferred clones were analyzed by SDS-PAGE to independently confirm expression (seeFIG. 30). The complete sequences of XTEN_AM923 and XTEN_AE912 were selected for further analysis.

• TABLE 22
  Preferred DNA Nucleotide Sequences for first 48 Amino Acid
  Residues of N-terminal XTEN-AM875 and XTEN-AE864

  			SEQ
  	XTEN		ID
  Clone Name	Modified	DNA Nucleotide Sequence	NO:
  LCW587_08	AM875	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCA	592
  		TCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCG
  		TCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCT
  		ACTGGCTCTCCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA
  LCW587_17	AM875	ATGGCTGAACCTGCTGGCTCTCCGACCTCTACTGAGGAAGGTACC	593
  		TCCCCTAGCGGCGAATCTTCTACTGCTCCAGGTACCTCTCCTAGC
  		GGCGAATCTTCTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCT
  		TCTACCGCACCAGGTACTTCTACTGAACCGTCTGAAGGCAGCGCA
  LCW588_14	AE864	ATGGCTGAACCTGCTGGCTCTCCAACCTCCACTGAGGAAGGTACC	594
  		CCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCCTT
  		CTGGTGCAACCGGCTCTCCAGGTGCTTCTCCGGGCACCAGCTCTA
  		CCGGTTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG
  LCW588_27	AE864	ATGGCTGAAACTGCTGGCTCTCCAACCTCCACTGAGGAAGGTGCA	595
  		TCCCCGGGCACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCG
  		TCTGGTGCTACCGGCTCTCCAGGTAGCTCTACCCCGTCTGGTGCT
  		ACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAG

Example 18Methods of Producing and Evaluating CFXTEN; XTEN-CF as Example
• A general schema for producing and evaluating CFXTEN compositions is presented inFIG. 33, and forms the basis for the general description of this Example. Using the disclosed methods and those known to one of ordinary skill in the art, together with guidance provided in the illustrative examples, a skilled artesian can create and evaluate a range of CFXTEN fusion proteins comprising, XTENs, CF and variants of CF known in the art. The Example is, therefore, to be construed as merely illustrative, and not limitative of the methods in any way whatsoever; numerous variations will be apparent to the ordinarily skilled artisan. In this Example, a CFXTEN of coagulation factor linked to an XTEN of the AE family of motifs is created.
• The general scheme for producing polynucleotides encoding XTEN is presented inFIGS. 31 and 32.FIG. 32 is a schematic flowchart of representative steps in the assembly of a XTEN polynucleotide construct in one of the embodiments of the invention.Individual oligonucleotides501 are annealed intosequence motifs502 such as a 12 amino acid motif (“12-mer”), which is subsequently ligated with an oligo containing BbsI, andKpnI restriction sites503. The motif libraries can be limited to specific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. In this case, the motifs of the AE family are used as the motif library, which are annealed to the 12-mer to create a “building block” length; e.g., a segment that encodes 36 amino acids. The gene encoding the XTEN sequence can be assembled by ligation and multimerization of the “building blocks” until the desired length of theXTEN gene504 is achieved. As illustrated inFIG. 32, the XTEN length in this case is 48 amino acid residues, but longer lengths can be achieved by this process. For example, multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. The XTEN gene can be cloned into a stuffer vector. In the example illustrated inFIG. 32, the vector can encode aFlag sequence506 followed by a stuffer sequence that is flanked by BsaI, BbsI, andKpnI sites507 and a CF gene (e.g., FVII)508, resulting in the gene encoding theCFXTEN500, which, in this case encodes the fusion protein in the configuration, N- to C-terminus, XTEN-FVII. As is apparent to one of ordinary skill in the art, the methods can be applied to create constructs in alternative configurations and with varying XTEN lengths.
• DNA sequences encoding CF can be conveniently obtained by standard procedures known in the art from a cDNA library prepared from an appropriate cellular source, from a genomic library, or may be created synthetically (e.g., automated nucleic acid synthesis) using DNA sequences obtained from publicly available databases, patents, or literature references. A gene or polynucleotide encoding the CF portion of the protein or its complement can be then be cloned into a construct, such as those described herein, which can be a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system. A second gene or polynucleotide coding for the XTEN portion or its complement (in the case ofFIG. 32 illustrated as an XTEN with 48 amino acid residues) can be genetically fused to the nucleotides encoding the terminus of the CF gene by cloning it into the construct adjacent and in frame with the gene coding for the CF, through a ligation or multimerization step. In this manner, a chimeric DNA molecule coding for (or complementary to) the CFXTEN fusion protein Re generated within the construct. Optionally, a gene encoding for a second XTEN are inserted and ligated in-frame to the nucleotides encoding the opposite terminus of the CFXTEN gene or can be inserted within the CF-encoding region. The construct can be designed in different configurations to encode the various permutations of the fusion partners as a monomeric polypeptide. For example, the gene can be created to encode the fusion protein in the order (N- to C-terminus): CF-XTEN; XTEN-CF; CF-XTEN-CF; XTEN-CF-XTEN; as well as multimers of the foregoing. Optionally, this chimeric DNA molecule is transferred or cloned into another construct that is a more appropriate expression vector. At this point, a host cell capable of expressing the chimeric DNA molecule is transformed with the chimeric DNA molecule. The vectors containing the DNA segments of interest can be transferred into an appropriate host cell by well-known methods, depending on the type of cellular host, as described supra.
• Host cells containing the XTEN-CF expression vector is cultured in conventional nutrient media modified as appropriate for activating the promoter. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. After expression of the fusion protein, culture broth is harvested and separated from the cell mass and the resulting crude extract retained for purification of the fusion protein.
• Gene expression are measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, gene expression is measured by immunological of fluorescent methods, such as immunohistochemical staining of cells to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against the CF sequence polypeptide using a synthetic peptide based on the sequences provided herein or against exogenous sequence fused to CF and encoding a specific antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).
• The CFXTEN polypeptide product is purified via methods known in the art. Procedures such as gel filtration, affinity purification, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography or gel electrophoresis are all techniques that may be used in the purification. Specific methods of purification are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor, ed., Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994).
• As illustrated inFIG. 33, the isolated CFXTEN fusion proteins would then be characterized for their chemical and activity properties. Isolated fusion protein is characterized, e.g., for sequence, purity, apparent molecular weight, solubility and stability using standard methods known in the art. The fusion protein meeting expected standards would then be evaluated for activity, which can be measured in vitro or in vivo by measuring one of the coagulation factor-associated parameters described herein, using one or more assays disclosed herein, or using the assays of the Examples or Table 40.
• In addition, the XTEN-CF fusion protein is administered to one or more animal species to determine standard pharmacokinetic parameters and pharmacodynamic properties, as described in Examples 30-33.
• By the iterative process of producing, expressing, and recovering CFXTEN constructs, followed by their characterization using methods disclosed herein or others known in the art, the CFXTEN compositions comprising CF and an XTEN can be produced and evaluated by one of ordinary skill in the art to confirm the expected properties such as enhanced solubility, enhanced stability, improved pharmacokinetics and reduced immunogenicity, leading to an overall enhanced therapeutic activity compared to the corresponding unfused CF. For those fusion proteins not possessing the desired properties, a different sequence can be constructed, expressed, isolated and evaluated by these methods in order to obtain a composition with such properties.
Example 19Construction of Expression Plasmids for FVII-XTEN
• Construction of FVII-TEV-XTEN 864 Expression Vectors
• The cloning vector containing the gene encoding FVII was purchased from OriGene (SC109205). PCR reactions were performed to abolish BbsI and BsaI restriction sites within the FVII coding region. The resulting FVII coding region was then amplified using primers that introduced NheI and TEV-BsaI sequences on the 5′ and 3′ end respectively. The digested FVII fragment was fused to BsaI/HindIII digested XTEN_AE864 fragment and inserted into NheI/HindIII digested pSecTag2C expression vector. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The final construct is pCW0647.001 which encodes the FVII-TEV-XTEN_AE864 protein (Table 23).
• Construction of FVII-XTEN 864 Expression Vectors
• FVII was amplified with pCW0647.001 as a template. The PCR primers introduced NheI and BsaI restriction enzyme recognition sequences on the 5′ and 3′ end respectively and deleted the TEV site. The NheI/BsaI digested FVII fragment was fused to BsaI/HindIII digested XTEN_AE864 fragment and inserted into NheI/HindIII digested pSecTag2C expression vector. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The final construct is pCW0645.001 which encodes the FVII-XTEN_AE864 protein (Table 23).
• Construction of Expression Vectors Encoding FVII-XTEN 864 Genes Using Millipore Plasmids
• Expression vector pCW0645.001 was digested with NheI and SalI. The resulting 4091 bp fragment included nucleotides that encode the FVII-XTEN_AE864 protein. This fragment was ligated with NheI/SalI digested CET1019-AS-puro, CET1019-HS-puro, SC AS-puro, or DC HS-puro (licensed from Millipore). These vectors feature a CMV promoter that lies upstream of the gene insertion site, and the CET1019 vectors also contain a UCOE element upstream of the promoter. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors were AC397 (pBC0013, SC AS puro-FVII-XTEN_AE864), AC402 (pBC0014, SC HS puro-FVII-XTEN_AE864), AC403 (pBC0015, CET1019 AS puro-FVII-XTEN_AE864), and AC404 (pBC0016, CET1019 HS puro-FVII-XTEN_AE864)
• Construction of Expression Vectors Encoding FVII-XTEN 288 Genes
• Expression vector pCW0645.001 was digested with BsaI and HindIII. The resulting 6400 bp fragment was ligated with BsaI/HindIII digested XTEN_AE288 fragment. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vector was pBC0019 (pSecTag2C-FVII-XTEN_AE288).
• Expression vector pBC0019 was digested with NheI and SalI. The resulting 2363 bp fragment included nucleotides that encode the FVII-XTEN_AE288 protein. This fragment was ligated with NheI/SalI digested CET1019-AS-puro, or CET1019-HS-puro (licensed from Millipore). These vectors feature a CMV promoter and a UCOE element that lie upstream of the gene insertion site. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors were AC405 (pBC0017, CET1019 AS puro-FVII-XTEN_AE288), and AC398 (pBC0018, CET1019 HS puro-FVII-XTEN_AE288) (Table 23).

• TABLE 23
  FVII amino acid and nucleic acid sequences

  		SEQ		SEQ
  		ID		ID
  Name	Amino Acid Sequence	NO:	Nucleic Acid Sequence	NO:

  FVII-TEV-	MVSQALRLLCLLLGLQ	596	ATGGTCTCCCAGGCCCTCAGGCTCCTCT	597
  XTEN_AE864,	GCLAAVFVTQEEAHGV		GCCTTCTGCTTGGGCTTCAGGGCTGCCT
  pCW0647.001	LHRRRRANAFLEELRP		GGCTGCAGTGTTCGTAACCCAGGAGGA
  	GSLERECKEEQCSFEEA		AGCCCACGGCGTCCTGCACCGGCGCCG
  	REIFKDAERTKLFWISY		GCGCGCCAACGCGTTCCTGGAGGAGCT
  	SDGDQCASSPCQNGGS		ACGGCCGGGCTCCCTGGAGAGGGAGTG
  	CKDQLQSYICFCLPAFE		CAAGGAGGAGCAGTGCTCCTTCGAGGA
  	GRNCETHKDDQLICVN		GGCCCGGGAGATCTTCAAGGACGCGGA
  	ENGGCEQYCSDHTGTK		GAGGACGAAGCTGTTCTGGATTTCTTAC
  	RSCRCHEGYSLLADGV		AGTGATGGGGACCAGTGTGCCTCAAGT
  	SCTPTVEYPCGKIPILEK		CCATGCCAGAATGGGGGCTCCTGCAAG
  	RNASKPQGRIVGGKVC		GACCAGCTCCAGTCCTATATCTGCTTCT
  	PKGECPWQVLLLVNGA		GCCTCCCTGCCTTCGAGGGCCGGAACTG
  	QLCGGTLINTIWVVSAA		TGAGACGCACAAGGATGACCAGCTGAT
  	HCFDKIKNWRNLIAVL		CTGTGTGAACGAGAACGGCGGCTGTGA
  	GEHDLSEHDGDEQSRR		GCAGTACTGCAGTGACCACACGGGCAC
  	VAQVIIPSTYVPGTTNH		CAAGCGCTCCTGTCGGTGCCACGAGGG
  	DIALLRLHQPVVLTDH		GTACTCTCTGCTGGCAGACGGGGTGTCC
  	VVPLCLPERTFSERTLA		TGCACACCCACAGTTGAATATCCATGTG
  	FVRFSLVSGWGQLLDR		GAAAAATACCTATTCTAGAAAAAAGAA
  	GATALELMVLNVPRLM		ATGCCAGCAAACCCCAAGGCCGAATTG
  	TQDCLQQSRKVGDSPNI		TGGGGGGCAAGGTGTGCCCCAAAGGGG
  	TEYMFCAGYSDGSKDS		AGTGTCCATGGCAGGTCCTGTTGTTGGT
  	CKGDSGGPHATHYRGT		GAATGGAGCTCAGTTGTGTGGGGGGAC
  	WYLTGIVSWGQGCATV		CCTGATCAACACCATCTGGGTGGTCTCC
  	GHFGVYTRVSQYIEWL		GCGGCCCACTGTTTCGACAAAATCAAG
  	QKLMRSEPRPGVLLRA		AACTGGAGGAACCTGATCGCGGTGCTG
  	PFPGPEGPSENLYFQGG		GGCGAGCACGACCTCAGCGAGCACGAC
  	SPGSPAGSPTSTEEGTSE		GGGGATGAGCAGAGCCGGCGGGTGGCG
  	SATPESGPGTSTEPSEGS		CAGGTCATCATCCCCAGCACGTACGTCC
  	APGSPAGSPTSTEEGTS		CGGGCACCACCAACCACGACATCGCGC
  	TEPSEGSAPGTSTEPSEG		TGCTCCGCCTGCACCAGCCCGTGGTCCT
  	SAPGTSESATPESGPGS		CACTGACCATGTGGTGCCCCTCTGCCTG
  	EPATSGSETPGSEPATS		CCCGAACGGACGTTCTCTGAGAGGACG
  	GSETPGSPAGSPTSTEE		CTGGCCTTCGTGCGCTTCTCATTGGTCA
  	GTSESATPESGPGTSTEP		GCGGCTGGGGCCAGCTGCTGGACCGTG
  	SEGSAPGTSTEPSEGSA		GCGCCACGGCCCTGGAGCTCATGGTCCT
  	PGSPAGSPTSTEEGTSTE		CAACGTGCCCCGGCTGATGACCCAGGA
  	PSEGSAPGTSTEPSEGS		CTGCCTGCAGCAGTCACGGAAGGTGGG
  	APGTSESATPESGPGTS		AGACTCCCCAAATATCACGGAGTACAT
  	TEPSEGSAPGTSESATPE		GTTCTGTGCCGGCTACTCGGATGGCAGC
  	SGPGSEPATSGSETPGT		AAGGACTCCTGCAAGGGGGACAGTGGA
  	STEPSEGSAPGTSTEPSE		GGCCCACATGCCACCCACTACCGGGGC
  	GSAPGTSESATPESGPG		ACGTGGTACCTGACGGGCATCGTCAGCT
  	TSESATPESGPGSPAGSP		GGGGCCAGGGCTGCGCAACCGTGGGCC
  	TSTEEGTSESATPESGP		ACTTTGGGGTGTACACCAGGGTCTCCCA
  	GSEPATSGSETPGTSES		GTACATCGAGTGGCTGCAAAAGCTCAT
  	ATPESGPGTSTEPSEGS		GCGCTCAGAGCCACGCCCAGGAGTCCT
  	APGTSTEPSEGSAPGTS		CCTGCGAGCCCCATTTCCCGGCCCAGAA
  	TEPSEGSAPGTSTEPSEG		GGCCCATCCGAAAATCTGTATTTTCAGG
  	SAPGTSTEPSEGSAPGT		GTGGGTCTCCAGGTTCTCCAGCCGGGTC
  	STEPSEGSAPGSPAGSPT		CCCAACTTCGACCGAGGAAGGGACCTC
  	STEEGTSTEPSEGSAPG		CGAGTCAGCTACCCCGGAGTCCGGTCCT
  	TSESATPESGPGSEPATS		GGCACCTCCACCGAACCATCGGAGGGC
  	GSETPGTSESATPESGP		AGCGCCCCTGGGAGCCCTGCCGGGAGC
  	GSEPATSGSETPGTSES		CCTACAAGCACCGAAGAGGGCACCAGT
  	ATPESGPGTSTEPSEGS		ACAGAGCCAAGTGAGGGGAGCGCCCCT
  	APGTSESATPESGPGSP		GGTACTAGTACTGAACCATCCGAGGGG
  	AGSPTSTEEGSPAGSPT		TCAGCTCCAGGCACGAGTGAGTCCGCTA
  	STEEGSPAGSPTSTEEG		CCCCCGAGAGCGGACCGGGCTCAGAGC
  	TSESATPESGPGTSTEPS		CCGCCACGAGTGGCAGTGAAACTCCAG
  	EGSAPGTSESATPESGP		GCTCAGAACCCGCCACTAGTGGGTCAG
  	GSEPATSGSETPGTSES		AGACTCCAGGCAGCCCTGCCGGATCCCC
  	ATPESGPGSEPATSGSE		TACGTCCACCGAGGAGGGAACATCTGA
  	TPGTSESATPESGPGTST		GTCCGCAACACCCGAATCCGGTCCAGG
  	EPSEGSAPGSPAGSPTST		CACCTCCACGGAACCTAGTGAAGGCTC
  	EEGTSESATPESGPGSEP		GGCACCAGGTACAAGCACCGAACCTAG
  	ATSGSETPGTSESATPES		CGAGGGCAGCGCTCCCGGCAGCCCTGC
  	GPGSPAGSPTSTEEGSP		CGGCAGCCCAACCTCAACTGAGGAGGG
  	AGSPTSTEEGTSTEPSE		CACCAGTACTGAGCCCAGCGAGGGATC
  	GSAPGTSESATPESGPG		AGCACCTGGCACCAGCACCGAACCTAG
  	TSESATPESGPGTSESAT		CGAGGGGAGCGCCCCTGGGACTAGCGA
  	PESGPGSEPATSGSETP		GTCAGCTACACCAGAGAGCGGGCCTGG
  	GSEPATSGSETPGSPAG		AACTTCTACCGAACCCAGTGAGGGATCC
  	SPTSTEEGTSTEPSEGSA		GCTCCAGGCACCTCCGAATCCGCAACCC
  	PGTSTEPSEGSAPGSEP		CCGAATCCGGACCTGGCTCAGAGCCCG
  	ATSGSETPGTSESATPES		CCACCAGCGGGAGCGAAACCCCTGGCA
  	GPGTSTEPSEGSAPGSSS		CATCCACCGAGCCTAGCGAAGGGTCCG
  			CACCCGGCACCAGTACAGAGCCTAGCG
  			AGGGATCAGCACCTGGCACCAGTGAAT
  			CTGCTACACCAGAGAGCGGCCCTGGAA
  			CCTCCGAGTCCGCTACCCCCGAGAGCGG
  			GCCAGGTTCTCCTGCTGGCTCCCCCACC
  			TCAACAGAAGAGGGGACAAGCGAAAGC
  			GCTACGCCTGAGAGTGGCCCTGGCTCTG
  			AGCCAGCCACCTCCGGCTCTGAAACCCC
  			TGGCACTAGTGAGTCTGCCACGCCTGAG
  			TCCGGACCCGGGACCTCTACTGAGCCCT
  			CGGAGGGGAGCGCTCCTGGCACGAGTA
  			CAGAACCTTCCGAAGGAAGTGCACCGG
  			GCACAAGCACCGAGCCTTCCGAAGGCT
  			CTGCTCCCGGAACCTCTACCGAACCCTC
  			TGAAGGGTCTGCACCCGGCACGAGCAC
  			CGAACCCAGCGAAGGGTCAGCGCCTGG
  			GACCTCAACAGAGCCCTCGGAAGGATC
  			AGCGCCTGGAAGCCCTGCAGGGAGTCC
  			AACTTCCACGGAAGAAGGAACGTCTAC
  			AGAGCCATCAGAGGGGTCCGCACCAGG
  			TACCAGCGAATCCGCTACTCCCGAATCT
  			GGCCCTGGGTCCGAACCTGCCACCTCCG
  			GCTCTGAAACTCCAGGGACCTCCGAATC
  			TGCCACACCCGAGAGCGGCCCTGGCTCC
  			GAGCCCGCAACATCTGGCAGCGAGACA
  			CCTGGCACCTCCGAGAGCGCAACACCC
  			GAGAGCGGCCCTGGCACCAGCACCGAG
  			CCATCCGAGGGATCCGCCCCAGGCACTT
  			CTGAGTCAGCCACACCCGAAAGCGGAC
  			CAGGATCACCCGCTGGCTCCCCCACCAG
  			TACCGAGGAGGGGTCCCCCGCTGGAAG
  			TCCAACAAGCACTGAGGAAGGGTCCCC
  			TGCCGGCTCCCCCACAAGTACCGAAGA
  			GGGCACAAGTGAGAGCGCCACTCCCGA
  			GTCCGGGCCTGGCACCAGCACAGAGCC
  			TTCCGAGGGGTCCGCACCAGGTACCTCA
  			GAGTCTGCTACCCCCGAGTCAGGGCCA
  			GGATCAGAGCCAGCCACCTCCGGGTCT
  			GAGACACCCGGGACTTCCGAGAGTGCC
  			ACCCCTGAGTCCGGACCCGGGTCCGAG
  			CCCGCCACTTCCGGCTCCGAAACTCCCG
  			GCACAAGCGAGAGCGCTACCCCAGAGT
  			CAGGACCAGGAACATCTACAGAGCCCT
  			CTGAAGGCTCCGCTCCAGGGTCCCCAGC
  			CGGCAGTCCCACTAGCACCGAGGAGGG
  			AACCTCTGAAAGCGCCACACCCGAATC
  			AGGGCCAGGGTCTGAGCCTGCTACCAG
  			CGGCAGCGAGACACCAGGCACCTCTGA
  			GTCCGCCACACCAGAGTCCGGACCCGG
  			ATCTCCCGCTGGGAGCCCCACCTCCACT
  			GAGGAGGGATCTCCTGCTGGCTCTCCAA
  			CATCTACTGAGGAAGGTACCTCAACCG
  			AGCCATCCGAGGGATCAGCTCCCGGCA
  			CCTCAGAGTCGGCAACCCCGGAGTCTG
  			GACCCGGAACTTCCGAAAGTGCCACAC
  			CAGAGTCCGGTCCCGGGACTTCAGAATC
  			AGCAACACCCGAGTCCGGCCCTGGGTCT
  			GAACCCGCCACAAGTGGTAGTGAGACA
  			CCAGGATCAGAACCTGCTACCTCAGGGT
  			CAGAGACACCCGGATCTCCGGCAGGCT
  			CACCAACCTCCACTGAGGAGGGCACCA
  			GCACAGAACCAAGCGAGGGCTCCGCAC
  			CCGGAACAAGCACTGAACCCAGTGAGG
  			GTTCAGCACCCGGCTCTGAGCCGGCCAC
  			AAGTGGCAGTGAGACACCCGGCACTTC
  			AGAGAGTGCCACCCCCGAGAGTGGCCC
  			AGGCACTAGTACCGAGCCCTCTGAAGG
  			CAGTGCGCCAGGTTCGTCTTCATAA
  FVII-	MVSQALRLLCLLLGLQ	598	ATGGTCTCCCAGGCCCTCAGGCTCCTCT	599
  XTEN_AE864,	GCLAAVFVTQEEAHGV		GCCTTCTGCTTGGGCTTCAGGGCTGCCT
  pCW0645.001	LHRRRRANAFLEELRP		GGCTGCAGTGTTCGTAACCCAGGAGGA
  	GSLERECKEEQCSFEEA		AGCCCACGGCGTCCTGCACCGGCGCCG
  	REIFKDAERTKLFWISY		GCGCGCCAACGCGTTCCTGGAGGAGCT
  	SDGDQCASSPCQNGGS		ACGGCCGGGCTCCCTGGAGAGGGAGTG
  	CKDQLQSYICFCLPAFE		CAAGGAGGAGCAGTGCTCCTTCGAGGA
  	GRNCETHKDDQLICVN		GGCCCGGGAGATCTTCAAGGACGCGGA
  	ENGGCEQYCSDHTGTK		GAGGACGAAGCTGTTCTGGATTTCTTAC
  	RSCRCHEGYSLLADGV		AGTGATGGGGACCAGTGTGCCTCAAGT
  	SCTPTVEYPCGKIPILEK		CCATGCCAGAATGGGGGCTCCTGCAAG
  	RNASKPQGRIVGGKVC		GACCAGCTCCAGTCCTATATCTGCTTCT
  	PKGECPWQVLLLVNGA		GCCTCCCTGCCTTCGAGGGCCGGAACTG
  	QLCGGTLINTIWVVSAA		TGAGACGCACAAGGATGACCAGCTGAT
  	HCFDKIKNWRNLIAVL		CTGTGTGAACGAGAACGGCGGCTGTGA
  	GEHDLSEHDGDEQSRR		GCAGTACTGCAGTGACCACACGGGCAC
  	VAQVIIPSTYVPGTTNH		CAAGCGCTCCTGTCGGTGCCACGAGGG
  	DIALLRLHQPVVLTDH		GTACTCTCTGCTGGCAGACGGGGTGTCC
  	VVPLCLPERTFSERTLA		TGCACACCCACAGTTGAATATCCATGTG
  	FVRFSLVSGWGQLLDR		GAAAAATACCTATTCTAGAAAAAAGAA
  	GATALELMVLNVPRLM		ATGCCAGCAAACCCCAAGGCCGAATTG
  	TQDCLQQSRKVGDSPNI		TGGGGGGCAAGGTGTGCCCCAAAGGGG
  	TEYMFCAGYSDGSKDS		AGTGTCCATGGCAGGTCCTGTTGTTGGT
  	CKGDSGGPHATHYRGT		GAATGGAGCTCAGTTGTGTGGGGGGAC
  	WYLTGIVSWGQGCATV		CCGATCAACACCATCTGGGTGGTCTCCG
  	GHFGVYTRVSQYIEWL		CGGCCCACTGTTTCGACAAAATCAAGA
  	QKLMRSEPRPGVLLRA		ACTGGAGGAACCTGATCGCGGTGCTGG
  	PFPGSPGSPAGSPTSTEE		GCGAGCACGACCTCAGCGAGCACGACG
  	GTSESATPESGPGTSTEP		GGGATGAGCAGAGCCGGCGGGTGGCGC
  	SEGSAPGSPAGSPTSTE		AGGTCATCATCCCCAGCACGTACGTCCC
  	EGTSTEPSEGSAPGTST		GGGCACCACCAACCACGACATCGCGCT
  	EPSEGSAPGTSESATPES		GCTCCGCCTGCACCAGCCCGTGGTCCTC
  	GPGSEPATSGSETPGSE		ACTGACCATGTGGTGCCCCTCTGCCTGC
  	PATSGSETPGSPAGSPTS		CCGAACGGACGTTCTCTGAGAGGACGC
  	TEEGTSESATPESGPGT		TGGCCTTCGTGCGCTTCTCATTGGTCAG
  	STEPSEGSAPGTSTEPSE		CGGCTGGGGCCAGCTGCTGGACCGTGG
  	GSAPGSPAGSPTSTEEG		CGCCACGGCCCTGGAGCTCATGGTCCTC
  	TSTEPSEGSAPGTSTEPS		AACGTGCCCCGGCTGATGACCCAGGAC
  	EGSAPGTSESATPESGP		TGCCTGCAGCAGTCACGGAAGGTGGGA
  	GTSTEPSEGSAPGTSES		GACTCCCCAAATATCACGGAGTACATGT
  	ATPESGPGSEPATSGSE		TCTGTGCCGGCTACTCGGATGGCAGCAA
  	TPGTSTEPSEGSAPGTST		GGACTCCTGCAAGGGGGACAGTGGAGG
  	EPSEGSAPGTSESATPES		CCCACATGCCACCCACTACCGGGGCAC
  	GPGTSESATPESGPGSP		GTGGTACCTGACGGGCATCGTCAGCTGG
  	AGSPTSTEEGTSESATP		GGCCAGGGCTGCGCAACCGTGGGCCAC
  	ESGPGSEPATSGSETPG		TTTGGGGTGTACACCAGGGTCTCCCAGT
  	TSESATPESGPGTSTEPS		ACATCGAGTGGCTGCAAAAGCTCATGC
  	EGSAPGTSTEPSEGSAP		GCTCAGAGCCACGCCCAGGAGTCCTCCT
  	GTSTEPSEGSAPGTSTEP		GCGAGCCCCATTTCCCGGAGGTAGCCCG
  	SEGSAPGTSTEPSEGSA		GCTGGCTCTCCTACCTCTACTGAGGAAG
  	PGTSTEPSEGSAPGSPA		GTACTTCTGAAAGCGCTACTCCTGAGTC
  	GSPTSTEEGTSTEPSEGS		TGGTCCAGGTACCTCTACTGAACCGTCC
  	APGTSESATPESGPGSE		GAAGGTAGCGCTCCAGGTAGCCCAGCA
  	PATSGSETPGTSESATPE		GGCTCTCCGACTTCCACTGAGGAAGGTA
  	SGPGSEPATSGSETPGT		CTTCTACTGAACCTTCCGAAGGCAGCGC
  	SESATPESGPGTSTEPSE		ACCAGGTACCTCTACTGAACCTTCTGAG
  	GSAPGTSESATPESGPG		GGCAGCGCTCCAGGTACTTCTGAAAGC
  	SPAGSPTSTEEGSPAGSP		GCTACCCCGGAATCTGGCCCAGGTAGC
  	TSTEEGSPAGSPTSTEE		GAACCGGCTACTTCTGGTTCTGAAACCC
  	GTSESATPESGPGTSTEP		CAGGTAGCGAACCGGCTACCTCCGGTTC
  	SEGSAPGTSESATPESG		TGAAACTCCAGGTAGCCCGGCAGGCTCT
  	PGSEPATSGSETPGTSES		CCGACCTCTACTGAGGAAGGTACTTCTG
  	ATPESGPGSEPATSGSE		AAAGCGCAACCCCGGAGTCCGGCCCAG
  	TPGTSESATPESGPGTST		GTACCTCTACCGAACCGTCTGAGGGCAG
  	EPSEGSAPGSPAGSPTST		CGCACCAGGTACTTCTACCGAACCGTCC
  	EEGTSESATPESGPGSEP		GAGGGTAGCGCACCAGGTAGCCCAGCA
  	ATSGSETPGTSESATPES		GGTTCTCCTACCTCCACCGAGGAAGGTA
  	GPGSPAGSPTSTEEGSP		CTTCTACCGAACCGTCCGAGGGTAGCGC
  	AGSPTSTEEGTSTEPSE		ACCAGGTACCTCTACTGAACCTTCTGAG
  	GSAPGTSESATPESGPG		GGCAGCGCTCCAGGTACTTCTGAAAGC
  	TSESATPESGPGTSESAT		GCTACCCCGGAGTCCGGTCCAGGTACTT
  	PESGPGSEPATSGSETP		CTACTGAACCGTCCGAAGGTAGCGCAC
  	GSEPATSGSETPGSPAG		CAGGTACTTCTGAAAGCGCAACCCCTGA
  	SPTSTEEGTSTEPSEGSA		ATCCGGTCCAGGTAGCGAACCGGCTACT
  	PGTSTEPSEGSAPGSEP		TCTGGCTCTGAGACTCCAGGTACTTCTA
  	ATSGSETPGTSESATPES		CCGAACCGTCCGAAGGTAGCGCACCAG
  	GPGTSTEPSEGSAPGSSS		GTACTTCTACTGAACCGTCTGAAGGTAG
  			CGCACCAGGTACTTCTGAAAGCGCAAC
  			CCCGGAATCCGGCCCAGGTACCTCTGAA
  			AGCGCAACCCCGGAGTCCGGCCCAGGT
  			AGCCCTGCTGGCTCTCCAACCTCCACCG
  			AAGAAGGTACCTCTGAAAGCGCAACCC
  			CTGAATCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCCAGGTAC
  			CTCTGAAAGCGCTACTCCGGAGTCTGGC
  			CCAGGTACCTCTACTGAACCGTCTGAGG
  			GTAGCGCTCCAGGTACTTCTACTGAACC
  			GTCCGAAGGTAGCGCACCAGGTACTTCT
  			ACCGAACCGTCCGAAGGCAGCGCTCCA
  			GGTACCTCTACTGAACCTTCCGAGGGCA
  			GCGCTCCAGGTACCTCTACCGAACCTTC
  			TGAAGGTAGCGCACCAGGTACTTCTACC
  			GAACCGTCCGAGGGTAGCGCACCAGGT
  			AGCCCAGCAGGTTCTCCTACCTCCACCG
  			AGGAAGGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCCAGGTAG
  			CGAACCTGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAACCCCG
  			GAATCTGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAGGTACCT
  			CTGAAAGCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCCGAGGGC
  			AGCGCACCAGGTACTTCTGAAAGCGCT
  			ACTCCTGAGTCCGGCCCAGGTAGCCCGG
  			CTGGCTCTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACTTCTACT
  			GAAGAAGGTAGCCCGGCAGGCTCTCCG
  			ACCTCTACTGAGGAAGGTACTTCTGAAA
  			GCGCAACCCCGGAGTCCGGCCCAGGTA
  			CCTCTACCGAACCGTCTGAGGGCAGCGC
  			ACCAGGTACCTCTGAAAGCGCAACTCCT
  			GAGTCTGGCCCAGGTAGCGAACCTGCT
  			ACCTCCGGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATCTGGTC
  			CAGGTAGCGAACCTGCAACCTCTGGCTC
  			TGAAACCCCAGGTACCTCTGAAAGCGCT
  			ACTCCTGAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGCACCAG
  			GTAGCCCTGCTGGCTCTCCAACCTCCAC
  			CGAAGAAGGTACCTCTGAAAGCGCAAC
  			CCCTGAATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACCCCAGGT
  			ACTTCTGAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTCTCCGAC
  			TTCCACCGAGGAAGGTAGCCCGGCTGG
  			CTCTCCAACTTCTACTGAAGAAGGTACT
  			TCTACCGAACCTTCCGAGGGCAGCGCAC
  			CAGGTACTTCTGAAAGCGCTACCCCTGA
  			GTCCGGCCCAGGTACTTCTGAAAGCGCT
  			ACTCCTGAATCCGGTCCAGGTACTTCTG
  			AAAGCGCTACCCCGGAATCTGGCCCAG
  			GTAGCGAACCGGCTACTTCTGGTTCTGA
  			AACCCCAGGTAGCGAACCGGCTACCTC
  			CGGTTCTGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGGAAGGTA
  			CTTCTACTGAACCTTCCGAAGGCAGCGC
  			ACCAGGTACCTCTACTGAACCTTCTGAG
  			GGCAGCGCTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAGGTACCT
  			CTGAAAGCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCCGAGGGC
  			AGCGCACCAGGTTAA
  FVII-	MVSQALRLLCLLLGLQ	600	ATGGTGTCCCAGGCCCTCAGGCTCCTCT	601
  XTEN_AE288,	GCLAAVFVTQEEAHGV		GCCTTCTGCTTGGGCTTCAGGGCTGCCT
  pBC0019	LHRRRRANAFLEELRP		GGCTGCAGTGTTCGTAACCCAGGAGGA
  	GSLERECKEEQCSFEEA		AGCCCACGGCGTCCTGCACCGGCGCCG
  	REIFKDAERTKLFWISY		GCGCGCCAACGCGTTCCTGGAGGAGCT
  	SDGDQCASSPCQNGGS		ACGGCCGGGCTCCCTGGAGAGGGAGTG
  	CKDQLQSYICFCLPAFE		CAAGGAGGAGCAGTGCTCCTTCGAGGA
  	GRNCETHKDDQLICVN		GGCCCGGGAGATCTTCAAGGACGCGGA
  	ENGGCEQYCSDHTGTK		GAGGACGAAGCTGTTCTGGATTTCTTAC
  	RSCRCHEGYSLLADGV		AGTGATGGGGACCAGTGTGCCTCAAGT
  	SCTPTVEYPCGKIPILEK		CCATGCCAGAATGGGGGCTCCTGCAAG
  	RNASKPQGRIVGGKVC		GACCAGCTCCAGTCCTATATCTGCTTCT
  	PKGECPWQVLLLVNGA		GCCTCCCTGCCTTCGAGGGCCGGAACTG
  	QLCGGTLINTIWVVSAA		TGAGACGCACAAGGATGACCAGCTGAT
  	HCFDKIKNWRNLIAVL		CTGTGTGAACGAGAACGGCGGCTGTGA
  	GEHDLSEHDGDEQSRR		GCAGTACTGCAGTGACCACACGGGCAC
  	VAQVIIPSTYVPGTTNH		CAAGCGCTCCTGTCGGTGCCACGAGGG
  	DIALLRLHQPVVLTDH		GTACTCTCTGCTGGCAGACGGGGTGTCC
  	VVPLCLPERTFSERTLA		TGCACACCCACAGTTGAATATCCATGTG
  	FVRFSLVSGWGQLLDR		GAAAAATACCTATTCTAGAAAAAAGAA
  	GATALELMVLNVPRLM		ATGCCAGCAAACCCCAAGGCCGAATTG
  	TQDCLQQSRKVGDSPNI		TGGGGGGCAAGGTGTGCCCCAAAGGGG
  	TEYMFCAGYSDGSKDS		AGTGTCCATGGCAGGTCCTGTTGTTGGT
  	CKGDSGGPHATHYRGT		GAATGGAGCTCAGTTGTGTGGGGGGAC
  	WYLTGIVSWGQGCATV		CCTGATCAACACCATCTGGGTGGTGTCC
  	GHFGVYTRVSQYIEWL		GCGGCCCACTGTTTCGACAAAATCAAG
  	QKLMRSEPRPGVLLRA		AACTGGAGAACCTGATCGCGGTGCTGG
  	PFPGSPGTSESATPESGP		GCGAGCACGACCTCAGCGAGCACGACG
  	GSEPATSGSETPGTSES		GGGATGAGCAGAGCCGGCGGGTGGCGC
  	ATPESGPGSEPATSGSE		AGGTCATCATCCCCAGCACGTACGTCCC
  	TPGTSESATPESGPGTST		GGGCACCACCAACCACGACATCGCGCT
  	EPSEGSAPGSPAGSPTST		GCTCCGCCTGCACCAGCCCGTGGTCCTC
  	EEGTSESATPESGPGSEP		ACTGACCATGTGGTGCCCCTCTGCCTGC
  	ATSGSETPGTSESATPES		CCGAACGGACGTTCTCTGAGAGGACGC
  	GPGSPAGSPTSTEEGSP		TGGCCTTCGTGCGCTTCTCATTGGTCAG
  	AGSPTSTEEGTSTEPSE		CGGCTGGGGCCAGCTGCTGGACCGTGG
  	GSAPGTSESATPESGPG		CGCCACGGCCCTGGAGCTCATGGTCCTC
  	TSESATPESGPGTSESAT		AACGTGCCCCGGCTGATGACCCAGGAC
  	PESGPGSEPATSGSETP		TGCCTGCAGCAGTCACGGAAGGTGGGA
  	GSEPATSGSETPGSPAG		GACTCCCCAAATATCACGGAGTACATGT
  	SPTSTEEGTSTEPSEGSA		TCTGTGCCGGCTACTCGGATGGCAGCAA
  	PGTSTEPSEGSAPGSEP		GGACTCCTGCAAGGGGGACAGTGGAGG
  	ATSGSETPGTSESATPES		CCCACATGCCACCCACTACCGGGGCAC
  	GPGTSTEPSEGSAPGSSS		GTGGTACCTGACGGGCATCGTCAGCTGG
  			GGCCAGGGCTGCGCAACCGTGGGCCAC
  			TTTGGGGTGTACACCAGGGTGTCCCAGT
  			ACATCGAGTGGCTGCAAAAGCTCATGC
  			GCTCAGAGCCACGCCCAGGAGTCCTCCT
  			GCGAGCCCCATTTCCCGGGTCTCCAGGT
  			ACCTCAGAGTCTGCTACCCCCGAGTCAG
  			GGCCAGGATCAGAGCCAGCCACCTCCG
  			GGTCTGAGACACCCGGGACTTCCGAGA
  			GTGCCACCCCTGAGTCCGGACCCGGGTC
  			CGAGCCCGCCACTTCCGGCTCCGAAACT
  			CCCGGCACAAGCGAGAGCGCTACCCCA
  			GAGTCAGGACCAGGAACATCTACAGAG
  			CCCTCTGAAGGCTCCGCTCCAGGGTCCC
  			CAGCCGGCAGTCCCACTAGCACCGAGG
  			AGGGAACCTCTGAAAGCGCCACACCCG
  			AATCAGGGCCAGGGTCTGAGCCTGCTA
  			CCAGCGGCAGCGAGACACCAGGCACCT
  			CTGAGTCCGCCACACCAGAGTCCGGAC
  			CCGGATCTCCCGCTGGGAGCCCCACCTC
  			CACTGAGGAGGGATCTCCTGCTGGCTCT
  			CCAACATCTACTGAGGAAGGTACCTCA
  			ACCGAGCCATCCGAGGGATCAGCTCCC
  			GGCACCTCAGAGTCGGCAACCCCGGAG
  			TCTGGACCCGGAACTTCCGAAAGTGCCA
  			CACCAGAGTCCGGTCCCGGGACTTCAG
  			AATCAGCAACACCCGAGTCCGGCCCTG
  			GGTCTGAACCCGCCACAAGTGGTAGTG
  			AGACACCAGGATCAGAACCTGCTACCT
  			CAGGGTCAGAGACACCCGGATCTCCGG
  			CAGGCTCACCAACCTCCACTGAGGAGG
  			GCACCAGCACAGAACCAAGCGAGGGCT
  			CCGCACCCGGAACAAGCACTGAACCCA
  			GTGAGGGTTCAGCACCCGGCTCTGAGCC
  			GGCCACAAGTGGCAGTGAGACACCCGG
  			CACTTCAGAGAGTGCCACCCCCGAGAG
  			TGGCCCAGGCACTAGTACCGAGCCCTCT
  			GAAGGCAGTGCGCCAGGTTCGTCTTCAT
  			AA

Example 20Construction of Expression Plasmids for FIX-XTEN
• Construction of FIX-XTEN 864 Genes and Vectors
• The cloning vector containing the gene encoding FIX was purchased from OriGene (SC126517). PCR reactions were performed to abolish two BbsI restriction sites within the FIX coding region. The resulting FIX coding region was then amplified using primers that introduced NheI and BsaI restriction enzyme recognition sequences on the 5′ and 3′ end respectively. The digested FIX fragment was fused to BsaI/HindIII digested XTEN_AM864, AE864, AF864, or AG864 fragments and inserted into NheI/HindIII digested pSecTag2C expression vector. The final constructs are AC282 (pCW0562, FIX-XTEN_AM864), AC283 (pCW0563, FIX-XTEN_AE864), pCW0564 (FIX-XTEN_AF864), and pCW0565 (FIX-XTEN_AG864) (Table 24).
• Construction of Expression Vectors for FIX Helper Genes
• The cloning vector containing the gene encoding PC5 was purchased from OriGene (SC310051). The PC5 coding region was amplified using primers that introduced NotI and BstBI restriction enzyme recognition sequences. The NotI/BstBI digested PC5 fragment was ligated with NotI/BstBI digested CET1019-HD-puro or DC-HD-puro vectors. Both CET1019-HD-puro and DC-HD-puro feature dual cassettes where a CMV promoter lies upstream of the gene insertion site, CET1019-HD-puro also contains a UCOE element upstream of the promoter. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors are pBC0037 (DC-HD-puro-PC5) and pBC0038 (CET1019 HD-puro-PC5).
• Construction of FIX-XTEN and PC5 Dual Expression Vectors
• The pBC0037 and pBC0038 constructs were digested with NheI and SalI and ligated with the NheI/SalI digested FIX-XTEN_AE864. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors were pBC0035 (DC-HD-puro-FIX-XTEN_AE864-PC5) and pBC0036 (CET1019-HD-puro-FIX-XTEN_AE864-PC5).

• TABLE 24
  FIX amino acid and nucleic acid sequences

  		SEQ		SEQ
  		ID		ID
  Name	Amino Acid Sequence	NO:	Nuclic Acid Sequences	NO:

  FIX-	MQRVNMIMAESPGLITI	602	ATGCAGCGCGTGAACATGATCAT	603
  XTEN_AM864,	CLLGYLLSAECTVFLDH		GGCAGAATCACCAGGCCTCATCA
  pCW0562	ENANKILNRPKRYNSG		CCATCTGCCTTTTAGGATATCTAC
  	KLEEFVQGNLERECME		TCAGTGCTGAATGTACAGTTTTTC
  	EKCSFEEAREVFENTER		TTGATCATGAAAACGCCAACAAA
  	TTEFWKQYVDGDQCES		ATTCTGAATCGGCCAAAGAGGTA
  	NPCLNGGSCKDDINSYE		TAATTCAGGTAAATTGGAAGAGT
  	CWCPFGFEGKNCELDV		TTGTTCAAGGGAACCTTGAGAGA
  	TCNIKNGRCEQFCKNS		GAATGTATGGAAGAAAAGTGTA
  	ADNKVVCSCTEGYRLA		GTTTTGAAGAAGCACGAGAAGTT
  	ENQKSCEPAVPFPCGRV		TTTGAAAACACTGAAAGAACAAC
  	SVSQTSKLTRAETVFPD		TGAATTTTGGAAGCAGTATGTTG
  	VDYVNSTEAETILDNIT		ATGGAGATCAGTGTGAGTCCAAT
  	QSTQSFNDFTRVVGGE		CCATGTTTAAATGGCGGCAGTTG
  	DAKPGQFPWQVVLNG		CAAGGATGACATTAATTCCTATG
  	KVDAFCGGSIVNEKWI		AATGTTGGTGTCCCTTTGGATTTG
  	VTAAHCVETGVKITVV		AAGGAAAGAACTGTGAATTAGAT
  	AGEHNIEETEHTEQKRN		GTAACATGTAACATTAAGAATGG
  	VIRIIPHHNYNAAINKY		CAGATGCGAGCAGTTTTGTAAAA
  	NHDIALLELDEPLVLNS		ATAGTGCTGATAACAAGGTGGTT
  	YVTPICIADKEYTNIFLK		TGCTCCTGTACTGAGGGATATCG
  	FGSGYVSGWGRVFHKG		ACTTGCAGAAAACCAGAAGTCCT
  	RSALVLQYLRVPLVDR		GTGAACCAGCAGTGCCATTTCCA
  	ATCLRSTKFTIYNNMFC		TGTGGAAGAGTTTCTGTTTCACA
  	AGFHEGGRDSCQGDSG		AACTTCTAAGCTCACCCGTGCTG
  	GPHVTEVEGTSFLTGIIS		AGACTGTTTTTCCTGATGTGGACT
  	WGEECAMKGKYGIYT		ATGTAAATTCTACTGAAGCTGAA
  	KVSRYVNWIKEKTKLT		ACCATTTTGGATAACATCACTCA
  	GGTSTEPSEGSAPGSEP		AAGCACCCAATCATTTAATGACT
  	ATSGSETPGSPAGSPTS		TCACTCGGGTTGTTGGTGGAGAA
  	TEEGSTSSTAESPGPGTS		GATGCCAAACCAGGTCAATTCCC
  	TPESGSASPGSTSESPSG		TTGGCAGGTTGTTTTGAATGGTA
  	TAPGSTSESPSGTAPGT		AAGTTGATGCATTCTGTGGAGGC
  	STPESGSASPGTSTPESG		TCTATCGTTAATGAAAAATGGAT
  	SASPGSEPATSGSETPG		TGTAACTGCTGCCCACTGTGTTG
  	TSESATPESGPGSPAGSP		AAACTGGTGTTAAAATTACAGTT
  	TSTEEGTSTEPSEGSAP		GTCGCAGGTGAACATAATATTGA
  	GTSESATPESGPGTSTEP		GGAGACAGAACATACAGAGCAA
  	SEGSAPGTSTEPSEGSA		AAGCGAAATGTGATTCGAATTAT
  	PGSPAGSPTSTEEGTSTE		TCCTCACCACAACTACAATGCAG
  	PSEGSAPGTSTEPSEGS		CTATTAATAAGTACAACCATGAC
  	APGTSESATPESGPGTS		ATTGCCCTTCTGGAACTGGACGA
  	ESATPESGPGTSTEPSEG		ACCCTTAGTGCTAAACAGCTACG
  	SAPGTSTEPSEGSAPGT		TTACACCTATTTGCATTGCTGACA
  	SESATPESGPGTSTEPSE		AGGAATACACGAACATCTTCCTC
  	GSAPGSEPATSGSETPG		AAATTTGGATCTGGCTATGTAAG
  	SPAGSPTSTEEGSSTPSG		TGGCTGGGGAAGAGTCTTCCACA
  	ATGSPGTPGSGTASSSP		AAGGGAGATCAGCTTTAGTTCTT
  	GSSTPSGATGSPGTSTE		CAGTACCTTAGAGTTCCACTTGTT
  	PSEGSAPGTSTEPSEGS		GACCGAGCCACATGTCTTCGATC
  	APGSEPATSGSETPGSP		TACAAAGTTCACCATCTATAACA
  	AGSPTSTEEGSPAGSPT		ACATGTTCTGTGCTGGCTTCCATG
  	STEEGTSTEPSEGSAPG		AAGGAGGTAGAGATTCATGTCAA
  	ASASGAPSTGGTSESAT		GGAGATAGTGGGGGACCCCATGT
  	PESGPGSPAGSPTSTEE		TACTGAAGTGGAAGGGACCAGTT
  	GSPAGSPTSTEEGSTSST		TCTTAACTGGAATTATTAGCTGG
  	AESPGPGSTSESPSGTAP		GGTGAAGAGTGTGCAATGAAAG
  	GTSPSGESSTAPGTPGS		GCAAATATGGAATATATACCAAG
  	GTASSSPGSSTPSGATG		GTATCCCGGTATGTCAACTGGAT
  	SPGSSPSASTGTGPGSEP		TAAGGAAAAAACAAAGCTCACT
  	ATSGSETPGTSESATPES		GGAGGTACTTCTACTGAACCGTC
  	GPGSEPATSGSETPGST		TGAAGGCAGCGCACCAGGTAGC
  	SSTAESPGPGSTSSTAES		GAACCGGCTACTTCCGGTTCTGA
  	PGPGTSPSGESSTAPGSE		AACCCCAGGTAGCCCAGCAGGTT
  	PATSGSETPGSEPATSG		CTCCAACTTCTACTGAAGAAGGT
  	SETPGTSTEPSEGSAPGS		TCTACCAGCTCTACCGCAGAATC
  	TSSTAESPGPGTSTPESG		TCCTGGTCCAGGTACCTCTACTCC
  	SASPGSTSESPSGTAPGT		GGAAAGCGGCTCTGCATCTCCAG
  	STEPSEGSAPGTSTEPSE		GTTCTACTAGCGAATCTCCTTCTG
  	GSAPGTSTEPSEGSAPG		GCACTGCACCAGGTTCTACTAGC
  	SSTPSGATGSPGSSPSAS		GAATCCCCGTCTGGTACTGCTCC
  	TGTGPGASPGTSSTGSP		AGGTACTTCTACTCCTGAAAGCG
  	GSEPATSGSETPGTSES		GTTCCGCTTCTCCAGGTACCTCTA
  	ATPESGPGSPAGSPTST		CTCCGGAAAGCGGTTCTGCATCT
  	EEGSSTPSGATGSPGSSP		CCAGGTAGCGAACCGGCAACCTC
  	SASTGTGPGASPGTSST		CGGCTCTGAAACCCCAGGTACCT
  	GSPGTSESATPESGPGT		CTGAAAGCGCTACTCCTGAATCC
  	STEPSEGSAPGTSTEPSE		GGCCCAGGTAGCCCGGCAGGTTC
  	GSAPG		TCCGACTTCCACTGAGGAAGGTA
  			CCTCTACTGAACCTTCTGAGGGC
  			AGCGCTCCAGGTACTTCTGAAAG
  			CGCTACCCCGGAGTCCGGTCCAG
  			GTACTTCTACTGAACCGTCCGAA
  			GGTAGCGCACCAGGTACTTCTAC
  			CGAACCGTCCGAGGGTAGCGCAC
  			CAGGTAGCCCAGCAGGTTCTCCT
  			ACCTCCACCGAGGAAGGTACTTC
  			TACCGAACCGTCCGAGGGTAGCG
  			CACCAGGTACTTCTACCGAACCT
  			TCCGAGGGCAGCGCACCAGGTAC
  			TTCTGAAAGCGCTACCCCTGAGT
  			CCGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAGG
  			TACCTCTACTGAACCTTCCGAAG
  			GCAGCGCTCCAGGTACCTCTACC
  			GAACCGTCCGAGGGCAGCGCACC
  			AGGTACTTCTGAAAGCGCAACCC
  			CTGAATCCGGTCCAGGTACTTCT
  			ACTGAACCTTCCGAAGGTAGCGC
  			TCCAGGTAGCGAACCTGCTACTT
  			CTGGTTCTGAAACCCCAGGTAGC
  			CCGGCTGGCTCTCCGACCTCCAC
  			CGAGGAAGGTAGCTCTACCCCGT
  			CTGGTGCTACTGGTTCTCCAGGT
  			ACTCCGGGCAGCGGTACTGCTTC
  			TTCCTCTCCAGGTAGCTCTACCCC
  			TTCTGGTGCTACTGGCTCTCCAG
  			GTACCTCTACCGAACCGTCCGAG
  			GGTAGCGCACCAGGTACCTCTAC
  			TGAACCGTCTGAGGGTAGCGCTC
  			CAGGTAGCGAACCGGCAACCTCC
  			GGTTCTGAAACTCCAGGTAGCCC
  			TGCTGGCTCTCCGACTTCTACTGA
  			GGAAGGTAGCCCGGCTGGTTCTC
  			CGACTTCTACTGAGGAAGGTACT
  			TCTACCGAACCTTCCGAAGGTAG
  			CGCTCCAGGTGCAAGCGCAAGCG
  			GCGCGCCAAGCACGGGAGGTACT
  			TCTGAAAGCGCTACTCCTGAGTC
  			CGGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTTC
  			TACTGAAGAAGGTTCTACCAGCT
  			CTACCGCTGAATCTCCTGGCCCA
  			GGTTCTACTAGCGAATCTCCGTC
  			TGGCACCGCACCAGGTACTTCCC
  			CTAGCGGTGAATCTTCTACTGCA
  			CCAGGTACCCCTGGCAGCGGTAC
  			CGCTTCTTCCTCTCCAGGTAGCTC
  			TACCCCGTCTGGTGCTACTGGCT
  			CTCCAGGTTCTAGCCCGTCTGCA
  			TCTACCGGTACCGGCCCAGGTAG
  			CGAACCGGCAACCTCCGGCTCTG
  			AAACTCCAGGTACTTCTGAAAGC
  			GCTACTCCGGAATCCGGCCCAGG
  			TAGCGAACCGGCTACTTCCGGCT
  			CTGAAACCCCAGGTTCCACCAGC
  			TCTACTGCAGAATCTCCGGGCCC
  			AGGTTCTACTAGCTCTACTGCAG
  			AATCTCCGGGTCCAGGTACTTCT
  			CCTAGCGGCGAATCTTCTACCGC
  			TCCAGGTAGCGAACCGGCAACCT
  			CTGGCTCTGAAACTCCAGGTAGC
  			GAACCTGCAACCTCCGGCTCTGA
  			AACCCCAGGTACTTCTACTGAAC
  			CTTCTGAGGGCAGCGCACCAGGT
  			TCTACCAGCTCTACCGCAGAATC
  			TCCTGGTCCAGGTACCTCTACTCC
  			GGAAAGCGGCTCTGCATCTCCAG
  			GTTCTACTAGCGAATCTCCTTCTG
  			GCACTGCACCAGGTACTTCTACC
  			GAACCGTCCGAAGGCAGCGCTCC
  			AGGTACCTCTACTGAACCTTCCG
  			AGGGCAGCGCTCCAGGTACCTCT
  			ACCGAACCTTCTGAAGGTAGCGC
  			ACCAGGTAGCTCTACTCCGTCTG
  			GTGCAACCGGCTCCCCAGGTTCT
  			AGCCCGTCTGCTTCCACTGGTAC
  			TGGCCCAGGTGCTTCCCCGGGCA
  			CCAGCTCTACTGGTTCTCCAGGT
  			AGCGAACCTGCTACCTCCGGTTC
  			TGAAACCCCAGGTACCTCTGAAA
  			GCGCAACTCCGGAGTCTGGTCCA
  			GGTAGCCCTGCAGGTTCTCCTAC
  			CTCCACTGAGGAAGGTAGCTCTA
  			CTCCGTCTGGTGCAACCGGCTCC
  			CCAGGTTCTAGCCCGTCTGCTTCC
  			ACTGGTACTGGCCCAGGTGCTTC
  			CCCGGGCACCAGCTCTACTGGTT
  			CTCCAGGTACCTCTGAAAGCGCT
  			ACTCCGGAGTCTGGCCCAGGTAC
  			CTCTACTGAACCGTCTGAGGGTA
  			GCGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAGG
  			TTAA
  FIX-	MQRVNMIMAESPGLITI	604	ATGCAGCGCGTGAACATGATCAT	605
  XTEN_AE864,	CLLGYLLSAECTVFLDH		GGCAGAATCACCAGGCCTCATCA
  pCW0563	ENANKILNRPKRYNSG		CCATCTGCCTTTTAGGATATCTAC
  	KLEEFVQGNLERECME		TCAGTGCTGAATGTACAGTTTTTC
  	EKCSFEEAREVFENTER		TTGATCATGAAAACGCCAACAAA
  	TTEFWKQYVDGDQCES		ATTCTGAATCGGCCAAAGAGGTA
  	NPCLNGGSCKDDINSYE		TAATTCAGGTAAATTGGAAGAGT
  	CWCPFGFEGKNCELDV		TTGTTCAAGGGAACCTTGAGAGA
  	TCNIKNGRCEQFCKNS		GAATGTATGGAAGAAAAGTGTA
  	ADNKVVCSCTEGYRLA		GTTTTGAAGAAGCACGAGAAGTT
  	ENQKSCEPAVPFPCGRV		TTTGAAAACACTGAAAGAACAAC
  	SVSQTSKLTRAETVFPD		TGAATTTTGGAAGCAGTATGTTG
  	VDYVNSTEAETILDNIT		ATGGAGATCAGTGTGAGTCCAAT
  	QSTQSFNDFTRVVGGE		CCATGTTTAAATGGCGGCAGTTG
  	DAKPGQFPWQVVLNG		CAAGGATGACATTAATTCCTATG
  	KVDAFCGGSIVNEKWI		AATGTTGGTGTCCCTTTGGATTTG
  	VTAAHCVETGVKITVV		AAGGAAAGAACTGTGAATTAGAT
  	AGEHNIEETEHTEQKRN		GTAACATGTAACATTAAGAATGG
  	VIRIIPHHNYNAAINKY		CAGATGCGAGCAGTTTTGTAAAA
  	NHDIALLELDEPLVLNS		ATAGTGCTGATAACAAGGTGGTT
  	YVTPICIADKEYTNIFLK		TGCTCCTGTACTGAGGGATATCG
  	FGSGYVSGWGRVFHKG		ACTTGCAGAAAACCAGAAGTCCT
  	RSALVLQYLRVPLVDR		GTGAACCAGCAGTGCCATTTCCA
  	ATCLRSTKFTIYNNMFC		TGTGGAAGAGTTTCTGTTTCACA
  	AGFHEGGRDSCQGDSG		AACTTCTAAGCTCACCCGTGCTG
  	GPHVTEVEGTSFLTGIIS		AGACTGTTTTTCCTGATGTGGACT
  	WGEECAMKGKYGIYT		ATGTAAATTCTACTGAAGCTGAA
  	KVSRYVNWIKEKTKLT		ACCATTTTGGATAACATCACTCA
  	GGSPAGSPTSTEEGTSE		AAGCACCCAATCATTTAATGACT
  	SATPESGPGTSTEPSEGS		TCACTCGGGTTGTTGGTGGAGAA
  	APGSPAGSPTSTEEGTS		GATGCCAAACCAGGTCAATTCCC
  	TEPSEGSAPGTSTEPSEG		TTGGCAGGTTGTTTTGAATGGTA
  	SAPGTSESATPESGPGS		AAGTTGATGCATTCTGTGGAGGC
  	EPATSGSETPGSEPATS		TCTATCGTTAATGAAAAATGGAT
  	GSETPGSPAGSPTSTEE		TGTAACTGCTGCCCACTGTGTTG
  	GTSESATPESGPGTSTEP		AAACTGGTGTTAAAATTACAGTT
  	SEGSAPGTSTEPSEGSA		GTCGCAGGTGAACATAATATTGA
  	PGSPAGSPTSTEEGTSTE		GGAGACAGAACATACAGAGCAA
  	PSEGSAPGTSTEPSEGS		AAGCGAAATGTGATTCGAATTAT
  	APGTSESATPESGPGTS		TCCTCACCACAACTACAATGCAG
  	TEPSEGSAPGTSESATPE		CTATTAATAAGTACAACCATGAC
  	SGPGSEPATSGSETPGT		ATTGCCCTTCTGGAACTGGACGA
  	STEPSEGSAPGTSTEPSE		ACCCTTAGTGCTAAACAGCTACG
  	GSAPGTSESATPESGPG		TTACACCTATTTGCATTGCTGACA
  	TSESATPESGPGSPAGSP		AGGAATACACGAACATCTTCCTC
  	TSTEEGTSESATPESGP		AAATTTGGATCTGGCTATGTAAG
  	GSEPATSGSETPGTSES		TGGCTGGGGAAGAGTCTTCCACA
  	ATPESGPGTSTEPSEGS		AAGGGAGATCAGCTTTAGTTCTT
  	APGTSTEPSEGSAPGTS		CAGTACCTTAGAGTTCCACTTGTT
  	TEPSEGSAPGTSTEPSEG		GACCGAGCCACATGTCTTCGATC
  	SAPGTSTEPSEGSAPGT		TACAAAGTTCACCATCTATAACA
  	STEPSEGSAPGSPAGSPT		ACATGTTCTGTGCTGGCTTCCATG
  	STEEGTSTEPSEGSAPG		AAGGAGGTAGAGATTCATGTCAA
  	TSESATPESGPGSEPATS		GGAGATAGTGGGGGACCCCATGT
  	GSETPGTSESATPESGP		TACTGAAGTGGAAGGGACCAGTT
  	GSEPATSGSETPGTSES		TCTTAACTGGAATTATTAGCTGG
  	ATPESGPGTSTEPSEGS		GGTGAAGAGTGTGCAATGAAAG
  	APGTSESATPESGPGSP		GCAAATATGGAATATATACCAAG
  	AGSPTSTEEGSPAGSPT		GTATCCCGGTATGTCAACTGGAT
  	STEEGSPAGSPTSTEEG		TAAGGAAAAAACAAAGCTCACT
  	TSESATPESGPGTSTEPS		GGAGGTAGCCCGGCTGGCTCTCC
  	EGSAPGTSESATPESGP		TACCTCTACTGAGGAAGGTACTT
  	GSEPATSGSETPGTSES		CTGAAAGCGCTACTCCTGAGTCT
  	ATPESGPGSEPATSGSE		GGTCCAGGTACCTCTACTGAACC
  	TPGTSESATPESGPGTST		GTCCGAAGGTAGCGCTCCAGGTA
  	EPSEGSAPGSPAGSPTST		GCCCAGCAGGCTCTCCGACTTCC
  	EEGTSESATPESGPGSEP		ACTGAGGAAGGTACTTCTACTGA
  	ATSGSETPGTSESATPES		ACCTTCCGAAGGCAGCGCACCAG
  	GPGSPAGSPTSTEEGSP		GTACCTCTACTGAACCTTCTGAG
  	AGSPTSTEEGTSTEPSE		GGCAGCGCTCCAGGTACTTCTGA
  	GSAPGTSESATPESGPG		AAGCGCTACCCCGGAATCTGGCC
  	TSESATPESGPGTSESAT		CAGGTAGCGAACCGGCTACTTCT
  	PESGPGSEPATSGSETP		GGTTCTGAAACCCCAGGTAGCGA
  	GSEPATSGSETPGSPAG		ACCGGCTACCTCCGGTTCTGAAA
  	SPTSTEEGTSTEPSEGSA		CTCCAGGTAGCCCGGCAGGCTCT
  	PGTSTEPSEGSAPGSEP		CCGACCTCTACTGAGGAAGGTAC
  	ATSGSETPGTSESATPES		TTCTGAAAGCGCAACCCCGGAGT
  	GPGTSTEPSEGSAPG		CCGGCCCAGGTACCTCTACCGAA
  			CCGTCTGAGGGCAGCGCACCAGG
  			TACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGCA
  			GGTTCTCCTACCTCCACCGAGGA
  			AGGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTACCTCT
  			ACTGAACCTTCTGAGGGCAGCGC
  			TCCAGGTACTTCTGAAAGCGCTA
  			CCCCGGAGTCCGGTCCAGGTACT
  			TCTACTGAACCGTCCGAAGGTAG
  			CGCACCAGGTACTTCTGAAAGCG
  			CAACCCCTGAATCCGGTCCAGGT
  			AGCGAACCGGCTACTTCTGGCTC
  			TGAGACTCCAGGTACTTCTACCG
  			AACCGTCCGAAGGTAGCGCACCA
  			GGTACTTCTACTGAACCGTCTGA
  			AGGTAGCGCACCAGGTACTTCTG
  			AAAGCGCAACCCCGGAATCCGGC
  			CCAGGTACCTCTGAAAGCGCAAC
  			CCCGGAGTCCGGCCCAGGTAGCC
  			CTGCTGGCTCTCCAACCTCCACC
  			GAAGAAGGTACCTCTGAAAGCGC
  			AACCCCTGAATCCGGCCCAGGTA
  			GCGAACCGGCAACCTCCGGTTCT
  			GAAACCCCAGGTACCTCTGAAAG
  			CGCTACTCCGGAGTCTGGCCCAG
  			GTACCTCTACTGAACCGTCTGAG
  			GGTAGCGCTCCAGGTACTTCTAC
  			TGAACCGTCCGAAGGTAGCGCAC
  			CAGGTACTTCTACCGAACCGTCC
  			GAAGGCAGCGCTCCAGGTACCTC
  			TACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACCT
  			TCTGAAGGTAGCGCACCAGGTAC
  			TTCTACCGAACCGTCCGAGGGTA
  			GCGCACCAGGTAGCCCAGCAGGT
  			TCTCCTACCTCCACCGAGGAAGG
  			TACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTACCTCTGAA
  			AGCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCCG
  			GCTCTGAGACTCCAGGTACCTCT
  			GAAAGCGCAACCCCGGAATCTGG
  			TCCAGGTAGCGAACCTGCAACCT
  			CTGGCTCTGAAACCCCAGGTACC
  			TCTGAAAGCGCTACTCCTGAATC
  			TGGCCCAGGTACTTCTACTGAAC
  			CGTCCGAGGGCAGCGCACCAGGT
  			ACTTCTGAAAGCGCTACTCCTGA
  			GTCCGGCCCAGGTAGCCCGGCTG
  			GCTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAAC
  			TTCTACTGAAGAAGGTAGCCCGG
  			CAGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAAC
  			CCCGGAGTCCGGCCCAGGTACCT
  			CTACCGAACCGTCTGAGGGCAGC
  			GCACCAGGTACCTCTGAAAGCGC
  			AACTCCTGAGTCTGGCCCAGGTA
  			GCGAACCTGCTACCTCCGGCTCT
  			GAGACTCCAGGTACCTCTGAAAG
  			CGCAACCCCGGAATCTGGTCCAG
  			GTAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTGA
  			AAGCGCTACTCCTGAATCTGGCC
  			CAGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGCC
  			CTGCTGGCTCTCCAACCTCCACC
  			GAAGAAGGTACCTCTGAAAGCGC
  			AACCCCTGAATCCGGCCCAGGTA
  			GCGAACCGGCAACCTCCGGTTCT
  			GAAACCCCAGGTACTTCTGAAAG
  			CGCTACTCCTGAGTCCGGCCCAG
  			GTAGCCCGGCTGGCTCTCCGACT
  			TCCACCGAGGAAGGTAGCCCGGC
  			TGGCTCTCCAACTTCTACTGAAG
  			AAGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTTC
  			TGAAAGCGCTACCCCTGAGTCCG
  			GCCCAGGTACTTCTGAAAGCGCT
  			ACTCCTGAATCCGGTCCAGGTAC
  			TTCTGAAAGCGCTACCCCGGAAT
  			CTGGCCCAGGTAGCGAACCGGCT
  			ACTTCTGGTTCTGAAACCCCAGG
  			TAGCGAACCGGCTACCTCCGGTT
  			CTGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGGA
  			AGGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTCT
  			ACTGAACCTTCTGAGGGCAGCGC
  			TCCAGGTAGCGAACCTGCAACCT
  			CTGGCTCTGAAACCCCAGGTACC
  			TCTGAAAGCGCTACTCCTGAATC
  			TGGCCCAGGTACTTCTACTGAAC
  			CGTCCGAGGGCAGCGCACCAGGT
  			TAA
  FIX-	MQRVNMIMAESPGLITI	606	ATGCAGCGCGTGAACATGATCAT	607
  XTEN_AF864,	CLLGYLLSAECTVFLDH		GGCAGAATCACCAGGCCTCATCA
  pCW0564	ENANKILNRPKRYNSG		CCATCTGCCTTTTAGGATATCTAC
  	KLEEFVQGNLERECME		TCAGTGCTGAATGTACAGTTTTTC
  	EKCSFEEAREVFENTER		TTGATCATGAAAACGCCAACAAA
  	TTEFWKQYVDGDQCES		ATTCTGAATCGGCCAAAGAGGTA
  	NPCLNGGSCKDDINSYE		TAATTCAGGTAAATTGGAAGAGT
  	CWCPFGFEGKNCELDV		TTGTTCAAGGGAACCTTGAGAGA
  	TCNIKNGRCEQFCKNS		GAATGTATGGAAGAAAAGTGTA
  	ADNKVVCSCTEGYRLA		GTTTTGAAGAAGCACGAGAAGTT
  	ENQKSCEPAVPFPCGRV		TTTGAAAACACTGAAAGAACAAC
  	SVSQTSKLTRAETVFPD		TGAATTTTGGAAGCAGTATGTTG
  	VDYVNSTEAETILDNIT		ATGGAGATCAGTGTGAGTCCAAT
  	QSTQSFNDFTRVVGGE		CCATGTTTAAATGGCGGCAGTTG
  	DAKPGQFPWQVVLNG		CAAGGATGACATTAATTCCTATG
  	KVDAFCGGSIVNEKWI		AATGTTGGTGTCCCTTTGGATTTG
  	VTAAHCVETGVKITVV		AAGGAAAGAACTGTGAATTAGAT
  	AGEHNIEETEHTEQKRN		GTAACATGTAACATTAAGAATGG
  	VIRIIPHHNYNAAINKY		CAGATGCGAGCAGTTTTGTAAAA
  	NHDIALLELDEPLVLNS		ATAGTGCTGATAACAAGGTGGTT
  	YVTPICIADKEYTNIFLK		TGCTCCTGTACTGAGGGATATCG
  	FGSGYVSGWGRVFHKG		ACTTGCAGAAAACCAGAAGTCCT
  	RSALVLQYLRVPLVDR		GTGAACCAGCAGTGCCATTTCCA
  	ATCLRSTKFTIYNNMFC		TGTGGAAGAGTTTCTGTTTCACA
  	AGFHEGGRDSCQGDSG		AACTTCTAAGCTCACCCGTGCTG
  	GPHVTEVEGTSFLTGIIS		AGACTGTTTTTCCTGATGTGGACT
  	WGEECAMKGKYGIYT		ATGTAAATTCTACTGAAGCTGAA
  	KVSRYVNWIKEKTKLT		ACCATTTTGGATAACATCACTCA
  	GGSTSESPSGTAPGTSPS		AAGCACCCAATCATTTAATGACT
  	GESSTAPGSTSESPSGT		TCACTCGGGTTGTTGGTGGAGAA
  	APGSTSESPSGTAPGTS		GATGCCAAACCAGGTCAATTCCC
  	TPESGSASPGTSTPESGS		TTGGCAGGTTGTTTTGAATGGTA
  	ASPGSTSESPSGTAPGST		AAGTTGATGCATTCTGTGGAGGC
  	SESPSGTAPGTSPSGESS		TCTATCGTTAATGAAAAATGGAT
  	TAPGSTSESPSGTAPGT		TGTAACTGCTGCCCACTGTGTTG
  	SPSGESSTAPGTSPSGES		AAACTGGTGTTAAAATTACAGTT
  	STAPGSTSSTAESPGPG		GTCGCAGGTGAACATAATATTGA
  	TSPSGESSTAPGTSPSGE		GGAGACAGAACATACAGAGCAA
  	SSTAPGSTSSTAESPGPG		AAGCGAAATGTGATTCGAATTAT
  	TSTPESGSASPGTSTPES		TCCTCACCACAACTACAATGCAG
  	GSASPGSTSESPSGTAP		CTATTAATAAGTACAACCATGAC
  	GSTSESPSGTAPGTSTPE		ATTGCCCTTCTGGAACTGGACGA
  	SGSASPGSTSSTAESPGP		ACCCTTAGTGCTAAACAGCTACG
  	GTSTPESGSASPGSTSES		TTACACCTATTTGCATTGCTGACA
  	PSGTAPGTSPSGESSTAP		AGGAATACACGAACATCTTCCTC
  	GSTSSTAESPGPGTSPSG		AAATTTGGATCTGGCTATGTAAG
  	ESSTAPGTSTPESGSASP		TGGCTGGGGAAGAGTCTTCCACA
  	GSTSSTAESPGPGSTSST		AAGGGAGATCAGCTTTAGTTCTT
  	AESPGPGSTSSTAESPGP		CAGTACCTTAGAGTTCCACTTGTT
  	GSTSSTAESPGPGTSPSG		GACCGAGCCACATGTCTTCGATC
  	ESSTAPGSTSESPSGTAP		TACAAAGTTCACCATCTATAACA
  	GSTSESPSGTAPGTSTPE		ACATGTTCTGTGCTGGCTTCCATG
  	SGPGGGGASASGAPST		AAGGAGGTAGAGATTCATGTCAA
  	GGGGSESPSGTAPGSTS		GGAGATAGTGGGGGACCCCATGT
  	ESPSGTAPGSTSESPSGT		TACTGAAGTGGAAGGGACCAGTT
  	APGSTSESPSGTAPGSTS		TCTTAACTGGAATTATTAGCTGG
  	ESPSGTAPGSTSESPSGT		GGTGAAGAGTGTGCAATGAAAG
  	APGTSTPESGSASPGTSP		GCAAATATGGAATATATACCAAG
  	SGESSTAPGTSPSGESST		GTATCCCGGTATGTCAACTGGAT
  	APGSTSSTAESPGPGTSP		TAAGGAAAAAACAAAGCTCACT
  	SGESSTAPGTSTPESGS		GGAGGTTCTACCAGCGAATCTCC
  	ASPGSTSESPSGTAPGST		TTCTGGCACCGCTCCAGGTACCT
  	SESPSGTAPGTSPSGESS		CTCCTAGCGGCGAATCTTCTACC
  	TAPGSTSESPSGTAPGT		GCTCCAGGTTCTACTAGCGAATC
  	STPESGSASPGTSTPESG		TCCTTCTGGCACTGCACCAGGTT
  	SASPGSTSESPSGTAPGT		CTACTAGCGAATCCCCGTCTGGT
  	STPESGSASPGSTSSTAE		ACTGCTCCAGGTACTTCTACTCCT
  	SPGPGSTSESPSGTAPGS		GAAAGCGGTTCCGCTTCTCCAGG
  	TSESPSGTAPGTSPSGES		TACCTCTACTCCGGAAAGCGGTT
  	STAPGSTSSTAESPGPG		CTGCATCTCCAGGTTCTACCAGC
  	TSPSGESSTAPGTSTPES		GAATCTCCTTCTGGCACCGCTCC
  	GSASPGTSPSGESSTAP		AGGTTCTACTAGCGAATCCCCGT
  	GTSPSGESSTAPGTSPSG		CTGGTACCGCACCAGGTACTTCT
  	ESSTAPGSTSSTAESPGP		CCTAGCGGCGAATCTTCTACCGC
  	GSTSSTAESPGPGTSPSG		ACCAGGTTCTACTAGCGAATCTC
  	ESSTAPGSSPSASTGTGP		CGTCTGGCACTGCTCCAGGTACT
  	GSSTPSGATGSPGSSTPS		TCTCCTAGCGGTGAATCTTCTACC
  	GATGSPG		GCTCCAGGTACTTCCCCTAGCGG
  			CGAATCTTCTACCGCTCCAGGTT
  			CTACTAGCTCTACTGCAGAATCT
  			CCGGGCCCAGGTACCTCTCCTAG
  			CGGTGAATCTTCTACCGCTCCAG
  			GTACTTCTCCGAGCGGTGAATCT
  			TCTACCGCTCCAGGTTCTACTAG
  			CTCTACTGCAGAATCTCCTGGCC
  			CAGGTACCTCTACTCCGGAAAGC
  			GGCTCTGCATCTCCAGGTACTTCT
  			ACCCCTGAAAGCGGTTCTGCATC
  			TCCAGGTTCTACTAGCGAATCTC
  			CTTCTGGCACTGCACCAGGTTCT
  			ACCAGCGAATCTCCGTCTGGCAC
  			TGCACCAGGTACCTCTACCCCTG
  			AAAGCGGTTCCGCTTCTCCAGGT
  			TCTACCAGCTCTACCGCAGAATC
  			TCCTGGTCCAGGTACCTCTACTCC
  			GGAAAGCGGCTCTGCATCTCCAG
  			GTTCTACTAGCGAATCTCCTTCTG
  			GCACTGCACCAGGTACTTCTCCG
  			AGCGGTGAATCTTCTACCGCACC
  			AGGTTCTACTAGCTCTACCGCTG
  			AATCTCCGGGCCCAGGTACTTCT
  			CCGAGCGGTGAATCTTCTACTGC
  			TCCAGGTACCTCTACTCCTGAAA
  			GCGGTTCTGCATCTCCAGGTTCC
  			ACTAGCTCTACCGCAGAATCTCC
  			GGGCCCAGGTTCTACTAGCTCTA
  			CTGCTGAATCTCCTGGCCCAGGT
  			TCTACTAGCTCTACTGCTGAATCT
  			CCGGGTCCAGGTTCTACCAGCTC
  			TACTGCTGAATCTCCTGGTCCAG
  			GTACCTCCCCGAGCGGTGAATCT
  			TCTACTGCACCAGGTTCTACTAG
  			CGAATCTCCTTCTGGCACTGCAC
  			CAGGTTCTACCAGCGAATCTCCG
  			TCTGGCACTGCACCAGGTACCTC
  			TACCCCTGAAAGCGGTCCGGGGG
  			GGGGGGGTGCAAGCGCAAGCGG
  			CGCGCCAAGCACGGGAGGGGGG
  			GGTAGCGAATCTCCTTCTGGTAC
  			CGCTCCAGGTTCTACCAGCGAAT
  			CCCCGTCTGGTACTGCTCCAGGT
  			TCTACCAGCGAATCTCCTTCTGGT
  			ACTGCACCAGGTTCTACTAGCGA
  			ATCTCCTTCTGGTACCGCTCCAG
  			GTTCTACCAGCGAATCCCCGTCT
  			GGTACTGCTCCAGGTTCTACCAG
  			CGAATCTCCTTCTGGTACTGCAC
  			CAGGTACTTCTACTCCGGAAAGC
  			GGTTCCGCATCTCCAGGTACTTCT
  			CCTAGCGGTGAATCTTCTACTGC
  			TCCAGGTACCTCTCCTAGCGGCG
  			AATCTTCTACTGCTCCAGGTTCTA
  			CCAGCTCTACTGCTGAATCTCCG
  			GGTCCAGGTACTTCCCCGAGCGG
  			TGAATCTTCTACTGCACCAGGTA
  			CTTCTACTCCGGAAAGCGGTTCC
  			GCTTCTCCAGGTTCTACCAGCGA
  			ATCTCCTTCTGGCACCGCTCCAG
  			GTTCTACTAGCGAATCCCCGTCT
  			GGTACCGCACCAGGTACTTCTCC
  			TAGCGGCGAATCTTCTACCGCAC
  			CAGGTTCTACTAGCGAATCCCCG
  			TCTGGTACCGCACCAGGTACTTC
  			TACCCCGGAAAGCGGCTCTGCTT
  			CTCCAGGTACTTCTACCCCGGAA
  			AGCGGCTCCGCATCTCCAGGTTC
  			TACTAGCGAATCTCCTTCTGGTA
  			CCGCTCCAGGTACTTCTACCCCT
  			GAAAGCGGCTCCGCTTCTCCAGG
  			TTCCACTAGCTCTACCGCTGAAT
  			CTCCGGGTCCAGGTTCTACCAGC
  			GAATCTCCTTCTGGCACCGCTCC
  			AGGTTCTACTAGCGAATCCCCGT
  			CTGGTACCGCACCAGGTACTTCT
  			CCTAGCGGCGAATCTTCTACCGC
  			ACCAGGTTCTACCAGCTCTACTG
  			CTGAATCTCCGGGTCCAGGTACT
  			TCCCCGAGCGGTGAATCTTCTAC
  			TGCACCAGGTACTTCTACTCCGG
  			AAAGCGGTTCCGCTTCTCCAGGT
  			ACCTCCCCTAGCGGCGAATCTTC
  			TACTGCTCCAGGTACCTCTCCTA
  			GCGGCGAATCTTCTACCGCTCCA
  			GGTACCTCCCCTAGCGGTGAATC
  			TTCTACCGCACCAGGTTCTACTA
  			GCTCTACTGCTGAATCTCCGGGT
  			CCAGGTTCTACCAGCTCTACTGC
  			TGAATCTCCTGGTCCAGGTACCT
  			CCCCGAGCGGTGAATCTTCTACT
  			GCACCAGGTTCTAGCCCTTCTGC
  			TTCCACCGGTACCGGCCCAGGTA
  			GCTCTACTCCGTCTGGTGCAACT
  			GGCTCTCCAGGTAGCTCTACTCC
  			GTCTGGTGCAACCGGCTCCCCAG
  			GTTAA
  FIX-	MQRVNMIMAESPGLITI	608	ATGCAGCGCGTGAACATGATCAT	609
  XTEN_AG864,	CLLGYLLSAECTVFLDH		GGCAGAATCACCAGGCCTCATCA
  pCW0565	ENANKILNRPKRYNSG		CCATCTGCCTTTTAGGATATCTAC
  	KLEEFVQGNLERECME		TCAGTGCTGAATGTACAGTTTTTC
  	EKCSFEEAREVFENTER		TTGATCATGAAAACGCCAACAAA
  	TTEFWKQYVDGDQCES		ATTCTGAATCGGCCAAAGAGGTA
  	NPCLNGGSCKDDINSYE		TAATTCAGGTAAATTGGAAGAGT
  	CWCPFGFEGKNCELDV		TTGTTCAAGGGAACCTTGAGAGA
  	TCNIKNGRCEQFCKNS		GAATGTATGGAAGAAAAGTGTA
  	ADNKVVCSCTEGYRLA		GTTTTGAAGAAGCACGAGAAGTT
  	ENQKSCEPAVPFPCGRV		TTTGAAAACACTGAAAGAACAAC
  	SVSQTSKLTRAETVFPD		TGAATTTTGGAAGCAGTATGTTG
  	VDYVNSTEAETILDNIT		ATGGAGATCAGTGTGAGTCCAAT
  	QSTQSFNDFTRVVGGE		CCATGTTTAAATGGCGGCAGTTG
  	DAKPGQFPWQVVLNG		CAAGGATGACATTAATTCCTATG
  	KVDAFCGGSIVNEKWI		AATGTTGGTGTCCCTTTGGATTTG
  	VTAAHCVETGVKITVV		AAGGAAAGAACTGTGAATTAGAT
  	AGEHNIEETEHTEQKRN		GTAACATGTAACATTAAGAATGG
  	VIRIIPHHNYNAAINKY		CAGATGCGAGCAGTTTTGTAAAA
  	NHDIALLELDEPLVLNS		ATAGTGCTGATAACAAGGTGGTT
  	YVTPICIADKEYTNIFLK		TGCTCCTGTACTGAGGGATATCG
  	FGSGYVSGWGRVFHKG		ACTTGCAGAAAACCAGAAGTCCT
  	RSALVLQYLRVPLVDR		GTGAACCAGCAGTGCCATTTCCA
  	ATCLRSTKFTIYNNMFC		TGTGGAAGAGTTTCTGTTTCACA
  	AGFHEGGRDSCQGDSG		AACTTCTAAGCTCACCCGTGCTG
  	GPHVTEVEGTSFLTGIIS		AGACTGTTTTTCCTGATGTGGACT
  	WGEECAMKGKYGIYT		ATGTAAATTCTACTGAAGCTGAA
  	KVSRYVNWIKEKTKLT		ACCATTTTGGATAACATCACTCA
  	GGASPGTSSTGSPGSSP		AAGCACCCAATCATTTAATGACT
  	SASTGTGPGSSPSASTG		TCACTCGGGTTGTTGGTGGAGAA
  	TGPGTPGSGTASSSPGS		GATGCCAAACCAGGTCAATTCCC
  	STPSGATGSPGSNPSAS		TTGGCAGGTTGTTTTGAATGGTA
  	TGTGPGASPGTSSTGSP		AAGTTGATGCATTCTGTGGAGGC
  	GTPGSGTASSSPGSSTPS		TCTATCGTTAATGAAAAATGGAT
  	GATGSPGTPGSGTASSS		TGTAACTGCTGCCCACTGTGTTG
  	PGASPGTSSTGSPGASP		AAACTGGTGTTAAAATTACAGTT
  	GTSSTGSPGTPGSGTAS		GTCGCAGGTGAACATAATATTGA
  	SSPGSSTPSGATGSPGA		GGAGACAGAACATACAGAGCAA
  	SPGTSSTGSPGTPGSGT		AAGCGAAATGTGATTCGAATTAT
  	ASSSPGSSTPSGATGSP		TCCTCACCACAACTACAATGCAG
  	GSNPSASTGTGPGSSPS		CTATTAATAAGTACAACCATGAC
  	ASTGTGPGSSTPSGATG		ATTGCCCTTCTGGAACTGGACGA
  	SPGSSTPSGATGSPGAS		ACCCTTAGTGCTAAACAGCTACG
  	PGTSSTGSPGASPGTSST		TTACACCTATTTGCATTGCTGACA
  	GSPGASPGTSSTGSPGT		AGGAATACACGAACATCTTCCTC
  	PGSGTASSSPGASPGTS		AAATTTGGATCTGGCTATGTAAG
  	STGSPGASPGTSSTGSP		TGGCTGGGGAAGAGTCTTCCACA
  	GASPGTSSTGSPGSSPS		AAGGGAGATCAGCTTTAGTTCTT
  	ASTGTGPGTPGSGTASS		CAGTACCTTAGAGTTCCACTTGTT
  	SPGASPGTSSTGSPGAS		GACCGAGCCACATGTCTTCGATC
  	PGTSSTGSPGASPGTSST		TACAAAGTTCACCATCTATAACA
  	GSPGSSTPSGATGSPGS		ACATGTTCTGTGCTGGCTTCCATG
  	STPSGATGSPGASPGTS		AAGGAGGTAGAGATTCATGTCAA
  	STGSPGTPGSGTASSSP		GGAGATAGTGGGGGACCCCATGT
  	GSSTPSGATGSPGSSTPS		TACTGAAGTGGAAGGGACCAGTT
  	GATGSPGSSTPSGATGS		TCTTAACTGGAATTATTAGCTGG
  	PGSSPSASTGTGPGASP		GGTGAAGAGTGTGCAATGAAAG
  	GTSSTGSPGASPGTSST		GCAAATATGGAATATATACCAAG
  	GSPGTPGSGTASSSPGA		GTATCCCGGTATGTCAACTGGAT
  	SPGTSSTGSPGASPGTSS		TAAGGAAAAAACAAAGCTCACT
  	TGSPGASPGTSSTGSPG		GGAGGTGCTTCCCCGGGCACCAG
  	ASPGTSSTGSPGTPGSG		CTCTACTGGTTCTCCAGGTTCTAG
  	TASSSPGSSTPSGATGSP		CCCGTCTGCTTCTACTGGTACTGG
  	GTPGSGTASSSPGSSTPS		TCCAGGTTCTAGCCCTTCTGCTTC
  	GATGSPGTPGSGTASSS		CACTGGTACTGGTCCAGGTACCC
  	PGSSTPSGATGSPGSSTP		CGGGTAGCGGTACCGCTTCTTCT
  	SGATGSPGSSPSASTGT		TCTCCAGGTAGCTCTACTCCGTCT
  	GPGSSPSASTGTGPGAS		GGTGCTACCGGCTCTCCAGGTTC
  	PGTSSTGSPGTPGSGTA		TAACCCTTCTGCATCCACCGGTA
  	SSSPGSSTPSGATGSPGS		CCGGCCCAGGTGCTTCTCCGGGC
  	SPSASTGTGPGSSPSAST		ACCAGCTCTACTGGTTCTCCAGG
  	GTGPGASPGTSSTGSPG		TACCCCGGGCAGCGGTACCGCAT
  	ASPGTSSTGSPGSSTPSG		CTTCTTCTCCAGGTAGCTCTACTC
  	ATGSPGSSPSASTGTGP		CTTCTGGTGCAACTGGTTCTCCA
  	GASPGTSSTGSPGSSPS		GGTACTCCTGGCAGCGGTACCGC
  	ASTGTGPGTPGSGTASS		TTCTTCTTCTCCAGGTGCTTCTCC
  	SPGSSTPSGATGSPGSST		TGGTACTAGCTCTACTGGTTCTCC
  	PSGATGSPGASPGTSST		AGGTGCTTCTCCGGGCACTAGCT
  	GSPG		CTACTGGTTCTCCAGGTACCCCG
  			GGTAGCGGTACTGCTTCTTCCTCT
  			CCAGGTAGCTCTACCCCTTCTGG
  			TGCAACCGGCTCTCCAGGTGCTT
  			CTCCGGGCACCAGCTCTACCGGT
  			TCTCCAGGTACCCCGGGTAGCGG
  			TACCGCTTCTTCTTCTCCAGGTAG
  			CTCTACTCCGTCTGGTGCTACCG
  			GCTCTCCAGGTTCTAACCCTTCTG
  			CATCCACCGGTACCGGCCCAGGT
  			TCTAGCCCTTCTGCTTCCACCGGT
  			ACTGGCCCAGGTAGCTCTACCCC
  			TTCTGGTGCTACCGGCTCCCCAG
  			GTAGCTCTACTCCTTCTGGTGCA
  			ACTGGCTCTCCAGGTGCATCTCC
  			GGGCACTAGCTCTACTGGTTCTC
  			CAGGTGCATCCCCTGGCACTAGC
  			TCTACTGGTTCTCCAGGTGCTTCT
  			CCTGGTACCAGCTCTACTGGTTCT
  			CCAGGTACTCCTGGCAGCGGTAC
  			CGCTTCTTCTTCTCCAGGTGCTTC
  			TCCTGGTACTAGCTCTACTGGTTC
  			TCCAGGTGCTTCTCCGGGCACTA
  			GCTCTACTGGTTCTCCAGGTGCTT
  			CCCCGGGCACTAGCTCTACCGGT
  			TCTCCAGGTTCTAGCCCTTCTGCA
  			TCTACTGGTACTGGCCCAGGTAC
  			TCCGGGCAGCGGTACTGCTTCTT
  			CCTCTCCAGGTGCATCTCCGGGC
  			ACTAGCTCTACTGGTTCTCCAGG
  			TGCATCCCCTGGCACTAGCTCTA
  			CTGGTTCTCCAGGTGCTTCTCCTG
  			GTACCAGCTCTACTGGTTCTCCA
  			GGTAGCTCTACTCCGTCTGGTGC
  			AACCGGTTCCCCAGGTAGCTCTA
  			CTCCTTCTGGTGCTACTGGCTCCC
  			CAGGTGCATCCCCTGGCACCAGC
  			TCTACCGGTTCTCCAGGTACCCC
  			GGGCAGCGGTACCGCATCTTCCT
  			CTCCAGGTAGCTCTACCCCGTCT
  			GGTGCTACCGGTTCCCCAGGTAG
  			CTCTACCCCGTCTGGTGCAACCG
  			GCTCCCCAGGTAGCTCTACTCCG
  			TCTGGTGCAACCGGCTCCCCAGG
  			TTCTAGCCCGTCTGCTTCCACTGG
  			TACTGGCCCAGGTGCTTCCCCGG
  			GCACCAGCTCTACTGGTTCTCCA
  			GGTGCATCCCCGGGTACCAGCTC
  			TACCGGTTCTCCAGGTACTCCTG
  			GCAGCGGTACTGCATCTTCCTCT
  			CCAGGTGCTTCTCCGGGCACCAG
  			CTCTACTGGTTCTCCAGGTGCATC
  			TCCGGGCACTAGCTCTACTGGTT
  			CTCCAGGTGCATCCCCTGGCACT
  			AGCTCTACTGGTTCTCCAGGTGC
  			TTCTCCTGGTACCAGCTCTACTGG
  			TTCTCCAGGTACCCCTGGTAGCG
  			GTACTGCTTCTTCCTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCTACC
  			GGTTCTCCAGGTACCCCGGGTAG
  			CGGTACCGCATCTTCTTCTCCAG
  			GTAGCTCTACCCCGTCTGGTGCT
  			ACTGGTTCTCCAGGTACTCCGGG
  			CAGCGGTACTGCTTCTTCCTCTCC
  			AGGTAGCTCTACCCCTTCTGGTG
  			CTACTGGCTCTCCAGGTAGCTCT
  			ACCCCGTCTGGTGCTACTGGCTC
  			CCCAGGTTCTAGCCCTTCTGCATC
  			CACCGGTACCGGTCCAGGTTCTA
  			GCCCGTCTGCATCTACTGGTACT
  			GGTCCAGGTGCATCCCCGGGCAC
  			TAGCTCTACCGGTTCTCCAGGTA
  			CTCCTGGTAGCGGTACTGCTTCTT
  			CTTCTCCAGGTAGCTCTACTCCTT
  			CTGGTGCTACTGGTTCTCCAGGTT
  			CTAGCCCTTCTGCATCCACCGGT
  			ACCGGCCCAGGTTCTAGCCCGTC
  			TGCTTCTACCGGTACTGGTCCAG
  			GTGCTTCTCCGGGTACTAGCTCT
  			ACTGGTTCTCCAGGTGCATCTCCT
  			GGTACTAGCTCTACTGGTTCTCC
  			AGGTAGCTCTACTCCGTCTGGTG
  			CAACCGGCTCTCCAGGTTCTAGC
  			CCTTCTGCATCTACCGGTACTGGT
  			CCAGGTGCATCCCCTGGTACCAG
  			CTCTACCGGTTCTCCAGGTTCTAG
  			CCCTTCTGCTTCTACCGGTACCGG
  			TCCAGGTACCCCTGGCAGCGGTA
  			CCGCATCTTCCTCTCCAGGTAGCT
  			CTACTCCGTCTGGTGCAACCGGT
  			TCCCCAGGTAGCTCTACTCCTTCT
  			GGTGCTACTGGCTCCCCAGGTGC
  			ATCCCCTGGCACCAGCTCTACCG
  			GTTCTCCAGGTTAA
  PC5, pBC0037,	MGWGSRCCCPGRLDLL	610	atgggctgggggagccgctgctgctgcccgggacgt	611
  pBC0038	CVLALLGGCLLPVCRT		ttggacctgctgtgcgtgctggcgctgctcgggggct
  	RVYTNHWAVKIAGGFP		gcctgctccccgtgtgtcggacgcgcgtctacaccaa
  	EANRIASKYGFINIGQIG		ccactgggcagtcaaaatcgccgggggcttcccgga
  	ALKDYYHFYHSRTIKRS		ggccaaccgtatcgccagcaagtacggattcatcaac
  	VISSRGTHSFISMEPKVE		ataggacagataggggccctgaaggactactaccact
  	WIQQQVVKKRTKRDY		tctaccatagcaggacgattaaaaggtcagttatctcg
  	DFSRAQSTYFNDPKWP		agcagagggacccacagtttcatttcaatggaaccaa
  	SMWYMHCSDNTHPCQ		aggtggaatggatccaacagcaagtggtaaaaaagc
  	SDMNIEGAWKRGYTG		ggacaaagagggattatgacttcagtcgtgcccagtct
  	KNIVVTILDDGIERTHP		acctatttcaatgatcccaagtggcccagcatgtggtat
  	DLMQNYDALASCDVN		atgcactgcagtgacaatacacatccctgccagtctga
  	GNDLDPMPRYDASNEN		catgaatatcgaaggagcctggaagagaggctacac
  	KHGTRCAGEVAAAAN		gggaaagaacattgtggtcactatcctggatgacgga
  	NSHCTVGIAFNAKIGGV		attgagagaacccatccagatctgatgcaaaactacg
  	RMLDGDVTDMVEAKS		atgctctggcaagttgcgacgtgaatgggaatgacttg
  	VSFNPQHVHIYSASWG		gacccaatgcctcgttatgatgcaagcaacgagaaca
  	PDDDGKTVDGPAPLTR		agcatgggactcgctgtgctggagaagtggcagccg
  	QAFENGVRMGRRGLGS		ctgcaaacaattcgcactgcacagtcggaattgctttc
  	VFVWASGNGGRSKDH		aacgccaagatcggaggagtgcgaatgctggacgg
  	CSCDGYTNSIYTISISST		agatgtcacggacatggttgaagcaaaatcagttagct
  	AESGKKPWYLEECSST		tcaacccccagcacgtgcacatttacagcgccagctg
  	LATTYSSGESYDKKIITT		gggcccggatgatgatggcaagactgtggacggacc
  	DLRQRCTDNHTGTSAS		agcccccctcacccggcaagcctttgaaaacggcgtt
  	APMAAGIIALALEANPF		agaatggggcggagaggcctcggctctgtgtttgtttg
  	LTWRDVQHVIVRTSRA		ggcatctggaaatggtggaaggagcaaagaccactg
  	GHLNANDWKTNAAGF		ctcctgtgatggctacaccaacagcatctacaccatct
  	KVSHLYGFGLMDAEA		ccatcagcagcactgcagaaagcggaaagaaacctt
  	MVMEAEKWTTVPRQH		ggtacctggaagagtgttcatccacgctggccacaac
  	VCVESTDRQIKTIRPNS		ctacagcagcggggagtcctacgataagaaaatcatc
  	AVRSIYKASGCSDNPNR		actacagatctgaggcagcgttgcacggacaaccac
  	HVNYLEHVVVRITITHP		actgggacgtcagcctcagcccccatggctgcaggc
  	RRGDLAIYLTSPSGTRS		atcattgcgctggccctggaagccaatccgtttctgac
  	QLLANRLFDHSMEGFK		ctggagagacgtacagcatgttattgtcaggacttccc
  	NWEFMTIHCWGERAA		gtgcgggacatttgaacgctaatgactggaaaaccaa
  	GDWVLEVYDTPSQLRN		tgctgctggttttaaggtgagccatctttatggatttgga
  	FKTPGKLKEWSLVLYG		ctgatggacgcagaagccatggtgatggaggcagag
  	TSVQPYSPTNEFPKVER		aagtggaccaccgttccccggcagcacgtgtgtgtgg
  	FRYSRVEDPTDDYGTE		agagcacagaccgacaaatcaagacaatccgcccta
  	DYAGPCDPECSEVGCD		acagtgcagtgcgctccatctacaaagcttcaggctg
  	GPGPDHCNDCLHYYYK		ctcggataaccccaaccgccatgtcaactacctggag
  	LKNNTRICVSSCPPGHY		cacgtcgttgtgcgcatcaccatcacccaccccagga
  	HADKKRCRKCAPNCES		gaggagacctggccatctacctgacctcgccctctgg
  	CFGSHGDQCMSCKYGY		aactaggtctcagcttttggccaacaggctatttgatca
  	FLNEETNSCVTHCPDGS		ctccatggaaggattcaaaaactgggagttcatgacc
  	YQDTKKNLCRKCSENC		attcattgctggggagaaagagctgctggtgactggg
  	KTCTEFHNCTECRDGLS		tccttgaagtttatgatactccctctcagctaaggaactt
  	LQGSRCSVSCEDGRYF		taagactccaggtaaattgaaagaatggtctttggtcct
  	NGQDCQPCHRFCATCA		ctacggcACCTCCGTGCAGCCATATT
  	GAGADGCINCTEGYFM		Caccaaccaatgaatttccgaaagtggaacggttccg
  	EDGRCVQSCSISYYFDH		ctatagccgagttgaagaccccacagacgactatggc
  	SSENGYKSCKKCDISCL		acagaggattatgcaggtccctgcgaccctgagtgca
  	TCNGPGFKNCTSCPSGY		gtgaggttggctgtgacgggccaggaccagaccact
  	LLDLGMCQMGAICKDA		gcaatgactgtttgcactactactacaagctgaaaaac
  	TEESWAEGGFCMLVKK		aataccaggatctgtgtctccagctgcccccctggcca
  	NNLCQRKVLQQLCCKT		ctaccacgccgacaagaagcgctgcaggaagtgtgc
  	CTFQG		ccccaactgtgagtcctgctttgggagccatggtgacc
  			aatgcatgtcctgcaaatatggatactttctgaatgaag
  			aaaccaacagctgtgttactcactgccctgatgggtca
  			tatcaggataccaagaaaaatctttgccggaaatgcag
  			tgaaaactgcaagacatgtactgaattccataactgtac
  			agaatgtagggatgggttaagcctgcagggatcccg
  			gtgctctgtctcctgtgaagatggacggtatttcaacgg
  			ccaggactgccagccctgccaccgcttctgcgccact
  			tgtgctggggcaggagctgatgggtgcattaactgca
  			cagagggctacttcatggaggatgggagatgcgtgc
  			agagctgtagtatcagctattactttgaccactcttcaga
  			gaatggatacaaatcctgcaaaaaatgtgatatcagtt
  			gtttgacgtgcaatggcccaggattcaagaactgtaca
  			agctgccctagtgggtatctcttagacttaggaatgtgt
  			caaatgggagccatttgcaaggatgcaacggaagag
  			tcctgggcggaaggaggcttctgtatgcttgtgaaaaa
  			gaacaatctgtgccaacggaaggttcttcaacaacttt
  			gctgcaaaacatgtacatttcaaggctga

Example 24Construction of FIX-/FXI/-XTEN
• Construction of FIX-TEV-XTEN_AE864 Genes and Vectors
• The cloning vector containing the gene encoding FIX was purchased from OriGene (SC126517). PCR reactions were performed to abolish two BbsI restriction sites within the FIX coding region. The resulting FIX coding region was then amplified using primers that introduced NheI and SfiI-TEV-BsaI sequences on the 5′ and 3′ end respectively. The digested FIX fragment was fused to BsaI/HindIII digested XTEN_AE864 fragment and inserted into NheI/HindIII digested pSecTag2C expression vector. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The final construct is pCW0648.001 which encodes the FIX-TEV-XTEN_AE864 protein (Table 25).
• Construction of FIX-/FXI/-XTEN_AE864 Genes and Vectors
• The TEV site was removed by digesting the pCW0648 expression vector with SfiI and BsaI. Oligos containing sequences that encode SfiI-KLTRAET-BsaI (‘KLTRAET’ disclosed as SEQ ID NO: 6), SfiI-DFTRVVG-BsaI (‘DFTRVVG’ disclosed as SEQ ID NO: 88), or SfiI-/FXI/-BsaI were annealed and ligated with the digested pCW0648 vector. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors encode FIX-KLTRAET-XTEN_AE864 (pCW0735) (‘KLTRAET’ disclosed as SEQ ID NO: 6), FIX-DFTRVVG-XTEN_AE864 (pCW0736) (‘DFTRVVG’ disclosed as SEQ ID NO: 88) and FIX-/FXI/-XTEN_AE864 (pCW0737).
• Construction of Expression Vectors Encoding FIX-/FXI/-XTEN_Ae864 Genes Using Millipore plasmids
• Expression vector pCW0735 was digested with NheI and SalI. The resulting 4181 bp fragment included nucleotides that encode the FIX-KLTRAET-XTEN_AE864 protein (‘KLTRAET’ disclosed as SEQ ID NO: 6). This fragment was ligated with NheI/SalI digested CET1019-HD-puro (Millipore) or DC HD-puro (Millipore). Both CET1019-HD-puro and DC-HD-puro feature dual cassettes where a CMV promoter lies upstream of the gene insertion site, CET1019-HD-puro also contains a UCOE element upstream of the promoter. The ligated DNA mixture was electroporated into XL1-Blue bacterial cells. Transformants were screened by DNA miniprep and the desired constructs were confirmed by DNA sequencing. The resulting expression vectors were pBC0033 (DC-HD-puro-FIX-KLTRAET-XTEN_AE864 (‘KLTRAET’ disclosed as SEQ ID NO: 6)) and pBC0034 (CET1019-HD-puro-FIX-KLTRAET-XTEN_AE864 (‘KLTRAET’ disclosed as SEQ ID NO: 6)) (Table 25).

• TABLE 25
  FIX-XTEN with cleavage sequence: amino acid and nucleic acid sequences

  		SEQ		SEQ
  		ID		ID
  Name	Ammo Acid Sequence	NO:	Nucleic Acid Sequences	NO:

  FIX-TEV-	MQRVNMIMAESPGLITI	612	atgcagcgcgtgaacatgatcatggcagaatcacc	613
  XTEN_AE864	CLLGYLLSAECTVFLDH		aggcctcatcaccatctgccttttaggatatctactc
  	ENANKILNRPKRYNSG		agtgctgaatgtacagtttttcttgatcatgaaaacg
  	KLEEFVQGNLERECME		ccaacaaaattctgaatcggccaaagaggtataatt
  	EKCSFEEAREVFENTER		caggtaaattggaagagtttgttcaagggaaccttg
  	TTEFWKQYVDGDQCES		agagagaatgtatggaagaaaagtgtagttttgaa
  	NPCLNGGSCKDDINSYE		gaagcacgagaagtttttgaaaacactgaaagaac
  	CWCPFGFEGKNCELDV		aactgaattttggaagcagtatgttgatggagatca
  	TCNIKNGRCEQFCKNS		gtgtgagtccaatccatgtttaaatggcggcagttg
  	ADNKVVCSCTEGYRLA		caaggatgacattaattcctatgaatgttggtgtccc
  	ENQKSCEPAVPFPCGRV		tttggatttgaaggaaagaactgtgaattagatgtaa
  	SVSQTSKLTRAETVFPD		catgtaacattaagaatggcagatgcgagcagtttt
  	VDYVNSTEAETILDNIT		gtaaaaatagtgctgataacaaggtggtttgctcct
  	QSTQSFNDFTRVVGGE		gtactgagggatatcgacttgcagaaaaccagaag
  	DAKPGQFPWQVVLNG		tcctgtgaaccagcagtgccatttccatgtggaaga
  	KVDAFCGGSIVNEKWI		gtttctgtttcacaaacttctaagctcacccgtgctga
  	VTAAHCVETGVKITVV		gactgtttttcctgatgtggactatgtaaattctactga
  	AGEHNIEETEHTEQKRN		agctgaaaccattttggataacatcactcaaagcac
  	VIRIIPHHNYNAAINKY		ccaatcatttaatgacttcactcgggttgttggtgga
  	NHDIALLELDEPLVLNS		gaagatgccaaaccaggtcaattcccttggcaggt
  	YVTPICIADKEYTNIFLK		tgttttgaatggtaaagttgatgcattctgtggaggct
  	FGSGYVSGWGRVFHKG		ctatcgttaatgaaaaatggattgtaactgctgccca
  	RSALVLQYLRVPLVDR		ctgtgttgaaactggtgttaaaattacagttgtcgca
  	ATCLRSTKFTIYNNMFC		ggtgaacataatattgaggagacagaacatacaga
  	AGFHEGGRDSCQGDSG		gcaaaagcgaaatgtgattcgaattattcctcacca
  	GPHVTEVEGTSFLTGIIS		caactacaatgcagctattaataagtacaaccatga
  	WGEECAMKGKYGIYT		cattgcccttctggaactggacgaacccttagtgct
  	KVSRYVNWIKEKTKLT		aaacagctacgttacacctatttgcattgctgacaag
  	GPEGPSENLYFQGGSPG		gaatacacgaacatcttcctcaaatttggatctggct
  	SPAGSPTSTEEGTSESAT		atgtaagtggctggggaagagtGttccacaaagg
  	PESGPGTSTEPSEGSAP		gagatcagctttagttcttcagtaccttagagttccac
  	GSPAGSPTSTEEGTSTEP		ttgttgaccgagccacatgtctAcgatctacaaagt
  	SEGSAPGTSTEPSEGSA		tcaccatctataacaacatgttctgtgctggcttccat
  	PGTSESATPESGPGSEP		gaaggaggtagagattcatgtcaaggagatagtg
  	ATSGSETPGSEPATSGS		ggggaccccatgttactgaagtggaagggaccag
  	ETPGSPAGSPTSTEEGTS		tttcttaactggaattattagctggggtgaagagtgt
  	ESATPESGPGTSTEPSEG		gcaatgaaaggcaaatatggaatatataccaaggt
  	SAPGTSTEPSEGSAPGSP		atcccggtatgtcaactggattaaggaaaaaacaa
  	AGSPTSTEEGTSTEPSE		agctcactGGCCCAGAAGGCCCAtcc
  	GSAPGTSTEPSEGSAPG		gaaaatctgtattttcagggtGGGTCTCCAG
  	TSESATPESGPGTSTEPS		GTTCTCCAGCCGGGTCCCCAAC
  	EGSAPGTSESATPESGP		TTCGACCGAGGAAGGGACCTC
  	GSEPATSGSETPGTSTEP		CGAGTCAGCTACCCCGGAGTCC
  	SEGSAPGTSTEPSEGSA		GGTCCTGGCACCTCCACCGAAC
  	PGTSESATPESGPGTSES		CATCGGAGGGCAGCGCCCCTG
  	ATPESGPGSPAGSPTST		GGAGCCCTGCCGGGAGCCCTA
  	EEGTSESATPESGPGSEP		CAAGCACCGAAGAGGGCACCA
  	ATSGSETPGTSESATPES		GTACAGAGCCAAGTGAGGGGA
  	GPGTSTEPSEGSAPGTS		GCGCCCCTGGTACTAGTACTGA
  	TEPSEGSAPGTSTEPSEG		ACCATCCGAGGGGTCAGCTCCA
  	SAPGTSTEPSEGSAPGT		GGCACGAGTGAGTCCGCTACCC
  	STEPSEGSAPGTSTEPSE		CCGAGAGCGGACCGGGCTCAG
  	GSAPGSPAGSPTSTEEG		AGCCCGCCACGAGTGGCAGTG
  	TSTEPSEGSAPGTSESAT		AAACTCCAGGCTCAGAACCCG
  	PESGPGSEPATSGSETP		CCACTAGTGGGTCAGAGACTCC
  	GTSESATPESGPGSEPA		AGGCAGCCCTGCCGGATCCCCT
  	TSGSETPGTSESATPESG		ACGTCCACCGAGGAGGGAACA
  	PGTSTEPSEGSAPGTSES		TCTGAGTCCGCAACACCCGAAT
  	ATPESGPGSPAGSPTST		CCGGTCCAGGCACCTCCACGGA
  	EEGSPAGSPTSTEEGSP		ACCTAGTGAAGGCTCGGCACC
  	AGSPTSTEEGTSESATP		AGGTACAAGCACCGAACCTAG
  	ESGPGTSTEPSEGSAPG		CGAGGGCAGCGCTCCCGGCAG
  	TSESATPESGPGSEPATS		CCCTGCCGGCAGCCCAACCTCA
  	GSETPGTSESATPESGP		ACTGAGGAGGGCACCAGTACT
  	GSEPATSGSETPGTSES		GAGCCCAGCGAGGGATCAGCA
  	ATPESGPGTSTEPSEGS		CCTGGCACCAGCACCGAACCTA
  	APGSPAGSPTSTEEGTS		GCGAGGGGAGCGCCCCTGGGA
  	ESATPESGPGSEPATSG		CTAGCGAGTCAGCTACACCAG
  	SETPGTSESATPESGPGS		AGAGCGGGCCTGGAACTTCTAC
  	PAGSPTSTEEGSPAGSP		CGAACCCAGTGAGGGATCCGC
  	TSTEEGTSTEPSEGSAP		TCCAGGCACCTCCGAATCCGCA
  	GTSESATPESGPGTSES		ACCCCCGAATCCGGACCTGGCT
  	ATPESGPGTSESATPES		CAGAGCCCGCCACCAGCGGGA
  	GPGSEPATSGSETPGSE		GCGAAACCCCTGGCACATCCAC
  	PATSGSETPGSPAGSPTS		CGAGCCTAGCGAAGGGTCCGC
  	TEEGTSTEPSEGSAPGT		ACCCGGCACCAGTACAGAGCC
  	STEPSEGSAPGSEPATS		TAGCGAGGGATCAGCACCTGG
  	GSETPGTSESATPESGP		CACCAGTGAATCTGCTACACCA
  	GTSTEPSEGSAPGSSS		GAGAGCGGCCCTGGAACCTCC
  			GAGTCCGCTACCCCCGAGAGC
  			GGGCCAGGTTCTCCTGCTGGCT
  			CCCCCACCTCAACAGAAGAGG
  			GGACAAGCGAAAGCGCTACGC
  			CTGAGAGTGGCCCTGGCTCTGA
  			GCCAGCCACCTCCGGCTCTGAA
  			ACCCCTGGCACTAGTGAGTCTG
  			CCACGCCTGAGTCCGGACCCGG
  			GACCTCTACTGAGCCCTCGGAG
  			GGGAGCGCTCCTGGCACGAGT
  			ACAGAACCTTCCGAAGGAAGT
  			GCACCGGGCACAAGCACCGAG
  			CCTTCCGAAGGCTCTGCTCCCG
  			GAACCTCTACCGAACCCTCTGA
  			AGGGTCTGCACCCGGCACGAG
  			CACCGAACCCAGCGAAGGGTC
  			AGCGCCTGGGACCTCAACAGA
  			GCCCTCGGAAGGATCAGCGCCT
  			GGAAGCCCTGCAGGGAGTCCA
  			ACTTCCACGGAAGAAGGAACG
  			TCTACAGAGCCATCAGAGGGG
  			TCCGCACCAGGTACCAGCGAAT
  			CCGCTACTCCCGAATCTGGCCC
  			TGGGTCCGAACCTGCCACCTCC
  			GGCTCTGAAACTCCAGGGACCT
  			CCGAATCTGCCACACCCGAGA
  			GCGGCCCTGGCTCCGAGCCCGC
  			AACATCTGGCAGCGAGACACC
  			TGGCACCTCCGAGAGCGCAAC
  			ACCCGAGAGCGGCCCTGGCAC
  			CAGCACCGAGCCATCCGAGGG
  			ATCCGCCCCAGGCACTTCTGAG
  			TCAGCCACACCCGAAAGCGGA
  			CCAGGATCACCCGCTGGCTCCC
  			CCACCAGTACCGAGGAGGGGT
  			CCCCCGCTGGAAGTCCAACAA
  			GCACTGAGGAAGGGTCCCCTG
  			CCGGCTCCCCCACAAGTACCGA
  			AGAGGGCACAAGTGAGAGCGC
  			CACTCCCGAGTCCGGGCCTGGC
  			ACCAGCACAGAGCCTTCCGAG
  			GGGTCCGCACCAGGTACCTCAG
  			AGTCTGCTACCCCCGAGTCAGG
  			GCCAGGATCAGAGCCAGCCAC
  			CTCCGGGTCTGAGACACCCGGG
  			ACTTCCGAGAGTGCCACCCCTG
  			AGTCCGGACCCGGGTCCGAGC
  			CCGCCACTTCCGGCTCCGAAAC
  			TCCCGGCACAAGCGAGAGCGC
  			TACCCCAGAGTCAGGACCAGG
  			AACATCTACAGAGCCCTCTGAA
  			GGCTCCGCTCCAGGGTCCCCAG
  			CCGGCAGTCCCACTAGCACCGA
  			GGAGGGAACCTCTGAAAGCGC
  			CACACCCGAATCAGGGCCAGG
  			GTCTGAGCCTGCTACCAGCGGC
  			AGCGAGACACCAGGCACCTCT
  			GAGTCCGCCACACCAGAGTCC
  			GGACCCGGATCTCCCGCTGGGA
  			GCCCCACCTCCACTGAGGAGG
  			GATCTCCTGCTGGCTCTCCAAC
  			ATCTACTGAGGAAGGTACCTCA
  			ACCGAGCCATCCGAGGGATCA
  			GCTCCCGGCACCTCAGAGTCGG
  			CAACCCCGGAGTCTGGACCCG
  			GAACTTCCGAAAGTGCCACACC
  			AGAGTCCGGTCCCGGGACTTCA
  			GAATCAGCAACACCCGAGTCC
  			GGCCCTGGGTCTGAACCCGCCA
  			CAAGTGGTAGTGAGACACCAG
  			GATCAGAACCTGCTACCTCAGG
  			GTCAGAGACACCCGGATCTCCG
  			GCAGGCTCACCAACCTCCACTG
  			AGGAGGGCACCAGCACAGAAC
  			CAAGCGAGGGCTCCGCACCCG
  			GAACAAGCACTGAACCCAGTG
  			AGGGTTCAGCACCCGGCTCTGA
  			GCCGGCCACAAGTGGCAGTGA
  			GACACCCGGCACTTCAGAGAG
  			TGCCACCCCCGAGAGTGGCCCA
  			GGCACTAGTACCGAGCCCTCTG
  			AAGGCAGTGCGCCAGGTTCGTC
  			TTCATAA
  FIX-	MQRVNMIMAESPGLITI	614	atgcagcgcgtgaacatgatcatggcagaatcacc	615
  KLTRAET-	CLLGYLLSAECTVFLDH		aggcctcatcaccatctgccttttaggatatctactc
  XTEN_AE864	ENANKILNRPKRYNSG		agtgctgaatgtacagtttttcttgatcatgaaaacg
  (‘KLTRAET’	KLEEFVQGNLERECME		ccaacaaaattctgaatcggccaaagaggtataatt
  disclosed as	EKCSFEEAREVFENTER		caggtaaattggaagagtttgttcaagggaaccttg
  SEQ ID NO: 6)	TTEFWKQYVDGDQCES		agagagaatgtatggaagaaaagtgtagttttgaa
  	NPCLNGGSCKDDINSYE		gaagcacgagaagtttttgaaaacactgaaagaac
  	CWCPFGFEGKNCELDV		aactgaattttggaagcagtatgttgatggagatca
  	TCNIKNGRCEQFCKNS		gtgtgagtccaatccatgtttaaatggcggcagttg
  	ADNKVVCSCTEGYRLA		caaggatgacattaattcctatgaatgttggtgtccc
  	ENQKSCEPAVPFPCGRV		tttggatttgaaggaaagaactgtgaattagatgtaa
  	SVSQTSKLTRAETVFPD		catgtaacattaagaatggcagatgcgagcagtttt
  	VDYVNSTEAETILDNIT		gtaaaaatagtgctgataacaaggtggtttgctcct
  	QSTQSFNDFTRVVGGE		gtactgagggatatcgacttgcagaaaaccagaag
  	DAKPGQFPWQVVLNG		tcctgtgaaccagcagtgccatttccatgtggaaga
  	KVDAFCGGSIVNEKWI		gtttctgtttcacaaacttctaagctcacccgtgctga
  	VTAAHCVETGVKITVV		gactgtttttcctgatgtggactatgtaaattctactga
  	AGEHNIEETEHTEQKRN		agctgaaaccattttggataacatcactcaaagcac
  	VIRIIPHHNYNAAINKY		ccaatcatttaatgacttcactcgggttgttggtgga
  	NHDIALLELDEPLVLNS		gaagatgccaaaccaggtcaattcccttggcaggt
  	YVTPICIADKEYTNIFLK		tgttttgaatggtaaagttgatgcattctgtggaggct
  	FGSGYVSGWGRVFHKG		ctatcgttaatgaaaaatggattgtaactgctgccca
  	RSALVLQYLRVPLVDR		ctgtgttgaaactggtgttaaaattacagttgtcgca
  	ATCLRSTKFTIYNNMFC		ggtgaacataatattgaggagacagaacatacaga
  	AGFHEGGRDSCQGDSG		gcaaaagcgaaatgtgattcgaattattcctcacca
  	GPHVTEVEGTSFLTGIIS		caactacaatgcagctattaataagtacaaccatga
  	WGEECAMKGKYGIYT		cattgcccttctggaactggacgaacccttagtgct
  	KVSRYVNWIKEKTKLT		aaacagctacgttacacctatttgcattgctgacaag
  	GPEGPSKLTRAETGSPG		gaatacacgaacatcttcctcaaatttggatctggct
  	SPAGSPTSTEEGTSESAT		atgtaagtggctggggaagagtGttccacaaagg
  	PESGPGTSTEPSEGSAP		gagatcagctttagttcttcagtaccttagagttccac
  	GSPAGSPTSTEEGTSTEP		ttgttgaccgagccacatgtctAcgatctacaaagt
  	SEGSAPGTSTEPSEGSA		tcaccatctataacaacatgttctgtgctggcttccat
  	PGTSESATPESGPGSEP		gaaggaggtagagattcatgtcaaggagatagtg
  	ATSGSETPGSEPATSGS		ggggaccccatgttactgaagtggaagggaccag
  	ETPGSPAGSPTSTEEGTS		tttcttaactggaattattagctggggtgaagagtgt
  	ESATPESGPGTSTEPSEG		gcaatgaaaggcaaatatggaatatataccaaggt
  	SAPGTSTEPSEGSAPGSP		atcccggtatgtcaactggattaaggaaaaaacaa
  	AGSPTSTEEGTSTEPSE		agctcactGGCCCAGAAGGCCCAtcc
  	GSAPGTSTEPSEGSAPG		aagctAacGcgtgcGgagacAGGGTCTC
  	TSESATPESGPGTSTEPS		CAGGTTCTCCAGCCGGGTCCCC
  	EGSAPGTSESATPESGP		AACTTCGACCGAGGAAGGGAC
  	GSEPATSGSETPGTSTEP		CTCCGAGTCAGCTACCCCGGAG
  	SEGSAPGTSTEPSEGSA		TCCGGTCCTGGCACCTCCACCG
  	PGTSESATPESGPGTSES		AACCATCGGAGGGCAGCGCCC
  	ATPESGPGSPAGSPTST		CTGGGAGCCCTGCCGGGAGCC
  	EEGTSESATPESGPGSEP		CTACAAGCACCGAAGAGGGCA
  	ATSGSETPGTSESATPES		CCAGTACAGAGCCAAGTGAGG
  	GPGTSTEPSEGSAPGTS		GGAGCGCCCCTGGTACTAGTAC
  	TEPSEGSAPGTSTEPSEG		TGAACCATCCGAGGGGTCAGCT
  	SAPGTSTEPSEGSAPGT		CCAGGCACGAGTGAGTCCGCT
  	STEPSEGSAPGTSTEPSE		ACCCCCGAGAGCGGACCGGGC
  	GSAPGSPAGSPTSTEEG		TCAGAGCCCGCCACGAGTGGC
  	TSTEPSEGSAPGTSESAT		AGTGAAACTCCAGGCTCAGAA
  	PESGPGSEPATSGSETP		CCCGCCACTAGTGGGTCAGAG
  	GTSESATPESGPGSEPA		ACTCCAGGCAGCCCTGCCGGAT
  	TSGSETPGTSESATPESG		CCCCTACGTCCACCGAGGAGG
  	PGTSTEPSEGSAPGTSES		GAACATCTGAGTCCGCAACACC
  	ATPESGPGSPAGSPTST		CGAATCCGGTCCAGGCACCTCC
  	EEGSPAGSPTSTEEGSP		ACGGAACCTAGTGAAGGCTCG
  	AGSPTSTEEGTSESATP		GCACCAGGTACAAGCACCGAA
  	ESGPGTSTEPSEGSAPG		CCTAGCGAGGGCAGCGCTCCC
  	TSESATPESGPGSEPATS		GGCAGCCCTGCCGGCAGCCCA
  	GSETPGTSESATPESGP		ACCTCAACTGAGGAGGGCACC
  	GSEPATSGSETPGTSES		AGTACTGAGCCCAGCGAGGGA
  	ATPESGPGTSTEPSEGS		TCAGCACCTGGCACCAGCACCG
  	APGSPAGSPTSTEEGTS		AACCTAGCGAGGGGAGCGCCC
  	ESATPESGPGSEPATSG		CTGGGACTAGCGAGTCAGCTAC
  	SETPGTSESATPESGPGS		ACCAGAGAGCGGGCCTGGAAC
  	PAGSPTSTEEGSPAGSP		TTCTACCGAACCCAGTGAGGGA
  	TSTEEGTSTEPSEGSAP		TCCGCTCCAGGCACCTCCGAAT
  	GTSESATPESGPGTSES		CCGCAACCCCCGAATCCGGACC
  	ATPESGPGTSESATPES		TGGCTCAGAGCCCGCCACCAGC
  	GPGSEPATSGSETPGSE		GGGAGCGAAACCCCTGGCACA
  	PATSGSETPGSPAGSPTS		TCCACCGAGCCTAGCGAAGGG
  	TEEGTSTEPSEGSAPGT		TCCGCACCCGGCACCAGTACAG
  	STEPSEGSAPGSEPATS		AGCCTAGCGAGGGATCAGCAC
  	GSETPGTSESATPESGP		CTGGCACCAGTGAATCTGCTAC
  	GTSTEPSEGSAPGSSS		ACCAGAGAGCGGCCCTGGAAC
  			CTCCGAGTCCGCTACCCCCGAG
  			AGCGGGCCAGGTTCTCCTGCTG
  			GCTCCCCCACCTCAACAGAAGA
  			GGGGACAAGCGAAAGCGCTAC
  			GCCTGAGAGTGGCCCTGGCTCT
  			GAGCCAGCCACCTCCGGCTCTG
  			AAACCCCTGGCACTAGTGAGTC
  			TGCCACGCCTGAGTCCGGACCC
  			GGGACCTCTACTGAGCCCTCGG
  			AGGGGAGCGCTCCTGGCACGA
  			GTACAGAACCTTCCGAAGGAA
  			GTGCACCGGGCACAAGCACCG
  			AGCCTTCCGAAGGCTCTGCTCC
  			CGGAACCTCTACCGAACCCTCT
  			GAAGGGTCTGCACCCGGCACG
  			AGCACCGAACCCAGCGAAGGG
  			TCAGCGCCTGGGACCTCAACAG
  			AGCCCTCGGAAGGATCAGCGC
  			CTGGAAGCCCTGCAGGGAGTC
  			CAACTTCCACGGAAGAAGGAA
  			CGTCTACAGAGCCATCAGAGG
  			GGTCCGCACCAGGTACCAGCG
  			AATCCGCTACTCCCGAATCTGG
  			CCCTGGGTCCGAACCTGCCACC
  			TCCGGCTCTGAAACTCCAGGGA
  			CCTCCGAATCTGCCACACCCGA
  			GAGCGGCCCTGGCTCCGAGCCC
  			GCAACATCTGGCAGCGAGACA
  			CCTGGCACCTCCGAGAGCGCA
  			ACACCCGAGAGCGGCCCTGGC
  			ACCAGCACCGAGCCATCCGAG
  			GGATCCGCCCCAGGCACTTCTG
  			AGTCAGCCACACCCGAAAGCG
  			GACCAGGATCACCCGCTGGCTC
  			CCCCACCAGTACCGAGGAGGG
  			GTCCCCCGCTGGAAGTCCAACA
  			AGCACTGAGGAAGGGTCCCCT
  			GCCGGCTCCCCCACAAGTACCG
  			AAGAGGGCACAAGTGAGAGCG
  			CCACTCCCGAGTCCGGGCCTGG
  			CACCAGCACAGAGCCTTCCGA
  			GGGGTCCGCACCAGGTACCTCA
  			GAGTCTGCTACCCCCGAGTCAG
  			GGCCAGGATCAGAGCCAGCCA
  			CCTCCGGGTCTGAGACACCCGG
  			GACTTCCGAGAGTGCCACCCCT
  			GAGTCCGGACCCGGGTCCGAG
  			CCCGCCACTTCCGGCTCCGAAA
  			CTCCCGGCACAAGCGAGAGCG
  			CTACCCCAGAGTCAGGACCAG
  			GAACATCTACAGAGCCCTCTGA
  			AGGCTCCGCTCCAGGGTCCCCA
  			GCCGGCAGTCCCACTAGCACCG
  			AGGAGGGAACCTCTGAAAGCG
  			CCACACCCGAATCAGGGCCAG
  			GGTCTGAGCCTGCTACCAGCGG
  			CAGCGAGACACCAGGCACCTC
  			TGAGTCCGCCACACCAGAGTCC
  			GGACCCGGATCTCCCGCTGGGA
  			GCCCCACCTCCACTGAGGAGG
  			GATCTCCTGCTGGCTCTCCAAC
  			ATCTACTGAGGAAGGTACCTCA
  			ACCGAGCCATCCGAGGGATCA
  			GCTCCCGGCACCTCAGAGTCGG
  			CAACCCCGGAGTCTGGACCCG
  			GAACTTCCGAAAGTGCCACACC
  			AGAGTCCGGTCCCGGGACTTCA
  			GAATCAGCAACACCCGAGTCC
  			GGCCCTGGGTCTGAACCCGCCA
  			CAAGTGGTAGTGAGACACCAG
  			GATCAGAACCTGCTACCTCAGG
  			GTCAGAGACACCCGGATCTCCG
  			GCAGGCTCACCAACCTCCACTG
  			AGGAGGGCACCAGCACAGAAC
  			CAAGCGAGGGCTCCGCACCCG
  			GAACAAGCACTGAACCCAGTG
  			AGGGTTCAGCACCCGGCTCTGA
  			GCCGGCCACAAGTGGCAGTGA
  			GACACCCGGCACTTCAGAGAG
  			TGCCACCCCCGAGAGTGGCCCA
  			GGCACTAGTACCGAGCCCTCTG
  			AAGGCAGTGCGCCAGGTTCGTC
  			TTCATAA
  FIX-	MQRVNMIMAESPGLITI	616	atgcagcgcgtgaacatgatcatggcagaatcacc	617
  DFTRVVG-	CLLGYLLSAECTVFLDH		aggcctcatcaccatctgccttttaggatatctactc
  XTEN_AE864	ENANKILNRPKRYNSG		agtgctgaatgtacagtttttcttgatcatgaaaacg
  	KLEEFVQGNLERECME		ccaacaaaattctgaatcggccaaagaggtataatt
  	EKCSFEEAREVFENTER		caggtaaattggaagagtttgttcaagggaaccttg
  	TTEFWKQYVDGDQCES		agagagaatgtatggaagaaaagtgtagttttgaa
  	NPCLNGGSCKDDINSYE		gaagcacgagaagtttttgaaaacactgaaagaac
  	CWCPFGFEGKNCELDV		aactgaattttggaagcagtatgttgatggagatca
  	TCNIKNGRCEQFCKNS		gtgtgagtccaatccatgtttaaatggcggcagttg
  	ADNKVVCSCTEGYRLA		caaggatgacattaattcctatgaatgttggtgtccc
  	ENQKSCEPAVPFPCGRV		tttggatttgaaggaaagaactgtgaattagatgtaa
  	SVSQTSKLTRAETVFPD		catgtaacattaagaatggcagatgcgagcagtttt
  	VDYVNSTEAETILDNIT		gtaaaaatagtgctgataacaaggtggtttgctcct
  	QSTQSFNDFTRVVGGE		gtactgagggatatcgacttgcagaaaaccagaag
  	DAKPGQFPWQVVLNG		tcctgtgaaccagcagtgccatttccatgtggaaga
  	KVDAFCGGSIVNEKWI		gtttctgtttcacaaacttctaagctcacccgtgctga
  	VTAAHCVETGVKITVV		gactgtttttcctgatgtggactatgtaaattctactga
  	AGEHNIEETEHTEQKRN		agctgaaaccattttggataacatcactcaaagcac
  	VIRIIPHHNYNAAINKY		ccaatcatttaatgacttcactcgggttgttggtgga
  	NHDIALLELDEPLVLNS		gaagatgccaaaccaggtcaattcccttggcaggt
  	YVTPICIADKEYTNIFLK		tgttttgaatggtaaagttgatgcattctgtggaggct
  	FGSGYVSGWGRVFHKG		ctatcgttaatgaaaaatggattgtaactgctgccca
  	RSALVLQYLRVPLVDR		ctgtgttgaaactggtgttaaaattacagttgtcgca
  	ATCLRSTKFTIYNNMFC		ggtgaacataatattgaggagacagaacatacaga
  	AGFHEGGRDSCQGDSG		gcaaaagcgaaatgtgattcgaattattcctcacca
  	GPHVTEVEGTSFLTGIIS		caactacaatgcagctattaataagtacaaccatga
  	WGEECAMKGKYGIYT		cattgcccttctggaactggacgaacccttagtgct
  	KVSRYVNWIKEKTKLT		aaacagctacgttacacctatttgcattgctgacaag
  	GPEGPSDFTRVVGGSPG		gaatacacgaacatcttcctcaaatttggatctggct
  	SPAGSPTSTEEGTSESAT		atgtaagtggctggggaagagtGttccacaaagg
  	PESGPGTSTEPSEGSAP		gagatcagctttagttcttcagtaccttagagttccac
  	GSPAGSPTSTEEGTSTEP		ttgttgaccgagccacatgtctAcgatctacaaagt
  	SEGSAPGTSTEPSEGSA		tcaccatctataacaacatgttctgtgctggcttccat
  	PGTSESATPESGPGSEP		gaaggaggtagagattcatgtcaaggagatagtg
  	ATSGSETPGSEPATSGS		ggggaccccatgttactgaagtggaagggaccag
  	ETPGSPAGSPTSTEEGTS		tttcttaactggaattattagctggggtgaagagtgt
  	ESATPESGPGTSTEPSEG		gcaatgaaaggcaaatatggaatatataccaaggt
  	SAPGTSTEPSEGSAPGSP		atcccggtatgtcaactggattaaggaaaaaacaa
  	AGSPTSTEEGTSTEPSE		agctcactGGCCCAGAAGGCCCAtcc
  	GSAPGTSTEPSEGSAPG		gacttcacAcgggtAgttggCGGGTCTCC
  	TSESATPESGPGTSTEPS		AGGTTCTCCAGCCGGGTCCCCA
  	EGSAPGTSESATPESGP		ACTTCGACCGAGGAAGGGACC
  	GSEPATSGSETPGTSTEP		TCCGAGTCAGCTACCCCGGAGT
  	SEGSAPGTSTEPSEGSA		CCGGTCCTGGCACCTCCACCGA
  	PGTSESATPESGPGTSES		ACCATCGGAGGGCAGCGCCCC
  	ATPESGPGSPAGSPTST		TGGGAGCCCTGCCGGGAGCCCT
  	EEGTSESATPESGPGSEP		ACAAGCACCGAAGAGGGCACC
  	ATSGSETPGTSESATPES		AGTACAGAGCCAAGTGAGGGG
  	GPGTSTEPSEGSAPGTS		AGCGCCCCTGGTACTAGTACTG
  	TEPSEGSAPGTSTEPSEG		AACCATCCGAGGGGTCAGCTCC
  	SAPGTSTEPSEGSAPGT		AGGCACGAGTGAGTCCGCTAC
  	STEPSEGSAPGTSTEPSE		CCCCGAGAGCGGACCGGGCTC
  	GSAPGSPAGSPTSTEEG		AGAGCCCGCCACGAGTGGCAG
  	TSTEPSEGSAPGTSESAT		TGAAACTCCAGGCTCAGAACCC
  	PESGPGSEPATSGSETP		GCCACTAGTGGGTCAGAGACTC
  	GTSESATPESGPGSEPA		CAGGCAGCCCTGCCGGATCCCC
  	TSGSETPGTSESATPESG		TACGTCCACCGAGGAGGGAAC
  	PGTSTEPSEGSAPGTSES		ATCTGAGTCCGCAACACCCGAA
  	ATPESGPGSPAGSPTST		TCCGGTCCAGGCACCTCCACGG
  	EEGSPAGSPTSTEEGSP		AACCTAGTGAAGGCTCGGCAC
  	AGSPTSTEEGTSESATP		CAGGTACAAGCACCGAACCTA
  	ESGPGTSTEPSEGSAPG		GCGAGGGCAGCGCTCCCGGCA
  	TSESATPESGPGSEPATS		GCCCTGCCGGCAGCCCAACCTC
  	GSETPGTSESATPESGP		AACTGAGGAGGGCACCAGTAC
  	GSEPATSGSETPGTSES		TGAGCCCAGCGAGGGATCAGC
  	ATPESGPGTSTEPSEGS		ACCTGGCACCAGCACCGAACCT
  	APGSPAGSPTSTEEGTS		AGCGAGGGGAGCGCCCCTGGG
  	ESATPESGPGSEPATSG		ACTAGCGAGTCAGCTACACCA
  	SETPGTSESATPESGPGS		GAGAGCGGGCCTGGAACTTCT
  	PAGSPTSTEEGSPAGSP		ACCGAACCCAGTGAGGGATCC
  	TSTEEGTSTEPSEGSAP		GCTCCAGGCACCTCCGAATCCG
  	GTSESATPESGPGTSES		CAACCCCCGAATCCGGACCTGG
  	ATPESGPGTSESATPES		CTCAGAGCCCGCCACCAGCGG
  	GPGSEPATSGSETPGSE		GAGCGAAACCCCTGGCACATC
  	PATSGSETPGSPAGSPTS		CACCGAGCCTAGCGAAGGGTC
  	TEEGTSTEPSEGSAPGT		CGCACCCGGCACCAGTACAGA
  	STEPSEGSAPGSEPATS		GCCTAGCGAGGGATCAGCACC
  	GSETPGTSESATPESGP		TGGCACCAGTGAATCTGCTACA
  	GTSTEPSEGSAPGSSS		CCAGAGAGCGGCCCTGGAACC
  			TCCGAGTCCGCTACCCCCGAGA
  			GCGGGCCAGGTTCTCCTGCTGG
  			CTCCCCCACCTCAACAGAAGAG
  			GGGACAAGCGAAAGCGCTACG
  			CCTGAGAGTGGCCCTGGCTCTG
  			AGCCAGCCACCTCCGGCTCTGA
  			AACCCCTGGCACTAGTGAGTCT
  			GCCACGCCTGAGTCCGGACCCG
  			GGACCTCTACTGAGCCCTCGGA
  			GGGGAGCGCTCCTGGCACGAG
  			TACAGAACCTTCCGAAGGAAG
  			TGCACCGGGCACAAGCACCGA
  			GCCTTCCGAAGGCTCTGCTCCC
  			GGAACCTCTACCGAACCCTCTG
  			AAGGGTCTGCACCCGGCACGA
  			GCACCGAACCCAGCGAAGGGT
  			CAGCGCCTGGGACCTCAACAG
  			AGCCCTCGGAAGGATCAGCGC
  			CTGGAAGCCCTGCAGGGAGTC
  			CAACTTCCACGGAAGAAGGAA
  			CGTCTACAGAGCCATCAGAGG
  			GGTCCGCACCAGGTACCAGCG
  			AATCCGCTACTCCCGAATCTGG
  			CCCTGGGTCCGAACCTGCCACC
  			TCCGGCTCTGAAACTCCAGGGA
  			CCTCCGAATCTGCCACACCCGA
  			GAGCGGCCCTGGCTCCGAGCCC
  			GCAACATCTGGCAGCGAGACA
  			CCTGGCACCTCCGAGAGCGCA
  			ACACCCGAGAGCGGCCCTGGC
  			ACCAGCACCGAGCCATCCGAG
  			GGATCCGCCCCAGGCACTTCTG
  			AGTCAGCCACACCCGAAAGCG
  			GACCAGGATCACCCGCTGGCTC
  			CCCCACCAGTACCGAGGAGGG
  			GTCCCCCGCTGGAAGTCCAACA
  			AGCACTGAGGAAGGGTCCCCT
  			GCCGGCTCCCCCACAAGTACCG
  			AAGAGGGCACAAGTGAGAGCG
  			CCACTCCCGAGTCCGGGCCTGG
  			CACCAGCACAGAGCCTTCCGA
  			GGGGTCCGCACCAGGTACCTCA
  			GAGTCTGCTACCCCCGAGTCAG
  			GGCCAGGATCAGAGCCAGCCA
  			CCTCCGGGTCTGAGACACCCGG
  			GACTTCCGAGAGTGCCACCCCT
  			GAGTCCGGACCCGGGTCCGAG
  			CCCGCCACTTCCGGCTCCGAAA
  			CTCCCGGCACAAGCGAGAGCG
  			CTACCCCAGAGTCAGGACCAG
  			GAACATCTACAGAGCCCTCTGA
  			AGGCTCCGCTCCAGGGTCCCCA
  			GCCGGCAGTCCCACTAGCACCG
  			AGGAGGGAACCTCTGAAAGCG
  			CCACACCCGAATCAGGGCCAG
  			GGTCTGAGCCTGCTACCAGCGG
  			CAGCGAGACACCAGGCACCTC
  			TGAGTCCGCCACACCAGAGTCC
  			GGACCCGGATCTCCCGCTGGGA
  			GCCCCACCTCCACTGAGGAGG
  			GATCTCCTGCTGGCTCTCCAAC
  			ATCTACTGAGGAAGGTACCTCA
  			ACCGAGCCATCCGAGGGATCA
  			GCTCCCGGCACCTCAGAGTCGG
  			CAACCCCGGAGTCTGGACCCG
  			GAACTTCCGAAAGTGCCACACC
  			AGAGTCCGGTCCCGGGACTTCA
  			GAATCAGCAACACCCGAGTCC
  			GGCCCTGGGTCTGAACCCGCCA
  			CAAGTGGTAGTGAGACACCAG
  			GATCAGAACCTGCTACCTCAGG
  			GTCAGAGACACCCGGATCTCCG
  			GCAGGCTCACCAACCTCCACTG
  			AGGAGGGCACCAGCACAGAAC
  			CAAGCGAGGGCTCCGCACCCG
  			GAACAAGCACTGAACCCAGTG
  			AGGGTTCAGCACCCGGCTCTGA
  			GCCGGCCACAAGTGGCAGTGA
  			GACACCCGGCACTTCAGAGAG
  			TGCCACCCCCGAGAGTGGCCCA
  			GGCACTAGTACCGAGCCCTCTG
  			AAGGCAGTGCGCCAGGTTCGTC
  			TTCATAA
  FIX-/FXI/-	MQRVNMIMAESPGLITI	618	atgcagcgcgtgaacatgatcatggcagaatcacc	619
  XTEN_AE864	CLLGYLLSAECTVFLDH		aggcctcatcaccatctgccttttaggatatctactc
  	ENANKILNRPKRYNSG		agtgctgaatgtacagtttttcttgatcatgaaaacg
  	KLEEFVQGNLERECME		ccaacaaaattctgaatcggccaaagaggtataatt
  	EKCSFEEAREVFENTER		caggtaaattggaagagtttgttcaagggaaccttg
  	TTEFWKQYVDGDQCES		agagagaatgtatggaagaaaagtgtagttttgaa
  	NPCLNGGSCKDDINSYE		gaagcacgagaagtttttgaaaacactgaaagaac
  	CWCPFGFEGKNCELDV		aactgaattttggaagcagtatgttgatggagatca
  	TCNIKNGRCEQFCKNS		gtgtgagtccaatccatgtttaaatggcggcagttg
  	ADNKVVCSCTEGYRLA		caaggatgacattaattcctatgaatgttggtgtccc
  	ENQKSCEPAVPFPCGRV		tttggatttgaaggaaagaactgtgaattagatgtaa
  	SVSQTSKLTRAETVFPD		catgtaacattaagaatggcagatgcgagcagtttt
  	VDYVNSTEAETILDNIT		gtaaaaatagtgctgataacaaggtggtttgctcct
  	QSTQSFNDFTRVVGGE		gtactgagggatatcgacttgcagaaaaccagaag
  	DAKPGQFPWQVVLNG		tcctgtgaaccagcagtgccatttccatgtggaaga
  	KVDAFCGGSIVNEKWI		gtttctgtttcacaaacttctaagctcacccgtgctga
  	VTAAHCVETGVKITVV		gactgtttttcctgatgtggactatgtaaattctactga
  	AGEHNIEETEHTEQKRN		agctgaaaccattttggataacatcactcaaagcac
  	VIRIIPHHNYNAAINKY		ccaatcatttaatgacttcactcgggttgttggtgga
  	NHDIALLELDEPLVLNS		gaagatgccaaaccaggtcaattcccttggcaggt
  	YVTPICIADKEYTNIFLK		tgttttgaatggtaaagttgatgcattctgtggaggct
  	FGSGYVSGWGRVFHKG		ctatcgttaatgaaaaatggattgtaactgctgccca
  	RSALVLQYLRVPLVDR		ctgtgttgaaactggtgttaaaattacagttgtcgca
  	ATCLRSTKFTIYNNMFC		ggtgaacataatattgaggagacagaacatacaga
  	AGFHEGGRDSCQGDSG		gcaaaagcgaaatgtgattcgaattattcctcacca
  	GPHVTEVEGTSFLTGIIS		caactacaatgcagctattaataagtacaaccatga
  	WGEECAMKGKYGIYT		cattgcccttctggaactggacgaacccttagtgct
  	KVSRYVNWIKEKTKLT		aaacagctacgttacacctatttgcattgctgacaag
  	GPEQTSKLTRAETVFPG		gaatacacgaacatcttcctcaaatttggatctggct
  	SPAGSPTSTEEGTSESAT		atgtaagtggctggggaagagtGttccacaaagg
  	PESGPGTSTEPSEGSAP		gagatcagctttagttcttcagtaccttagagttccac
  	GSPAGSPTSTEEGTSTEP		ttgttgaccgagccacatgtctAcgatctacaaagt
  	SEGSAPGTSTEPSEGSA		tcaccatctataacaacatgttctgtgctggcttccat
  	PGTSESATPESGPGSEP		gaaggaggtagagattcatgtcaaggagatagtg
  	ATSGSETPGSEPATSGS		ggggaccccatgttactgaagtggaagggaccag
  	ETPGSPAGSPTSTEEGTS		tttcttaactggaattattagctggggtgaagagtgt
  	ESATPESGPGTSTEPSEG		gcaatgaaaggcaaatatggaatatataccaaggt
  	SAPGTSTEPSEGSAPGSP		atcccggtatgtcaactggattaaggaaaaaacaa
  	AGSPTSTEEGTSTEPSE		agctcactGGCCCAGAAcaaacAtctaag
  	GSAPGTSTEPSEGSAPG		ctAacGcgtgcGgagacAgtAtttccaGGT
  	TSESATPESGPGTSTEPS		TCTCCAGCCGGGTCCCCAACTT
  	EGSAPGTSESATPESGP		CGACCGAGGAAGGGACCTCCG
  	GSEPATSGSETPGTSTEP		AGTCAGCTACCCCGGAGTCCGG
  	SEGSAPGTSTEPSEGSA		TCCTGGCACCTCCACCGAACCA
  	PGTSESATPESGPGTSES		TCGGAGGGCAGCGCCCCTGGG
  	ATPESGPGSPAGSPTST		AGCCCTGCCGGGAGCCCTACA
  	EEGTSESATPESGPGSEP		AGCACCGAAGAGGGCACCAGT
  	ATSGSETPGTSESATPES		ACAGAGCCAAGTGAGGGGAGC
  	GPGTSTEPSEGSAPGTS		GCCCCTGGTACTAGTACTGAAC
  	TEPSEGSAPGTSTEPSEG		CATCCGAGGGGTCAGCTCCAG
  	SAPGTSTEPSEGSAPGT		GCACGAGTGAGTCCGCTACCCC
  	STEPSEGSAPGTSTEPSE		CGAGAGCGGACCGGGCTCAGA
  	GSAPGSPAGSPTSTEEG		GCCCGCCACGAGTGGCAGTGA
  	TSTEPSEGSAPGTSESAT		AACTCCAGGCTCAGAACCCGCC
  	PESGPGSEPATSGSETP		ACTAGTGGGTCAGAGACTCCA
  	GTSESATPESGPGSEPA		GGCAGCCCTGCCGGATCCCCTA
  	TSGSETPGTSESATPESG		CGTCCACCGAGGAGGGAACAT
  	PGTSTEPSEGSAPGTSES		CTGAGTCCGCAACACCCGAATC
  	ATPESGPGSPAGSPTST		CGGTCCAGGCACCTCCACGGA
  	EEGSPAGSPTSTEEGSP		ACCTAGTGAAGGCTCGGCACC
  	AGSPTSTEEGTSESATP		AGGTACAAGCACCGAACCTAG
  	ESGPGTSTEPSEGSAPG		CGAGGGCAGCGCTCCCGGCAG
  	TSESATPESGPGSEPATS		CCCTGCCGGCAGCCCAACCTCA
  	GSETPGTSESATPESGP		ACTGAGGAGGGCACCAGTACT
  	GSEPATSGSETPGTSES		GAGCCCAGCGAGGGATCAGCA
  	ATPESGPGTSTEPSEGS		CCTGGCACCAGCACCGAACCTA
  	APGSPAGSPTSTEEGTS		GCGAGGGGAGCGCCCCTGGGA
  	ESATPESGPGSEPATSG		CTAGCGAGTCAGCTACACCAG
  	SETPGTSESATPESGPGS		AGAGCGGGCCTGGAACTTCTAC
  	PAGSPTSTEEGSPAGSP		CGAACCCAGTGAGGGATCCGC
  	TSTEEGTSTEPSEGSAP		TCCAGGCACCTCCGAATCCGCA
  	GTSESATPESGPGTSES		ACCCCCGAATCCGGACCTGGCT
  	ATPESGPGTSESATPES		CAGAGCCCGCCACCAGCGGGA
  	GPGSEPATSGSETPGSE		GCGAAACCCCTGGCACATCCAC
  	PATSGSETPGSPAGSPTS		CGAGCCTAGCGAAGGGTCCGC
  	TEEGTSTEPSEGSAPGT		ACCCGGCACCAGTACAGAGCC
  	STEPSEGSAPGSEPATS		TAGCGAGGGATCAGCACCTGG
  	GSETPGTSESATPESGP		CACCAGTGAATCTGCTACACCA
  	GTSTEPSEGSAPGSSS		GAGAGCGGCCCTGGAACCTCC
  			GAGTCCGCTACCCCCGAGAGC
  			GGGCCAGGTTCTCCTGCTGGCT
  			CCCCCACCTCAACAGAAGAGG
  			GGACAAGCGAAAGCGCTACGC
  			CTGAGAGTGGCCCTGGCTCTGA
  			GCCAGCCACCTCCGGCTCTGAA
  			ACCCCTGGCACTAGTGAGTCTG
  			CCACGCCTGAGTCCGGACCCGG
  			GACCTCTACTGAGCCCTCGGAG
  			GGGAGCGCTCCTGGCACGAGT
  			ACAGAACCTTCCGAAGGAAGT
  			GCACCGGGCACAAGCACCGAG
  			CCTTCCGAAGGCTCTGCTCCCG
  			GAACCTCTACCGAACCCTCTGA
  			AGGGTCTGCACCCGGCACGAG
  			CACCGAACCCAGCGAAGGGTC
  			AGCGCCTGGGACCTCAACAGA
  			GCCCTCGGAAGGATCAGCGCCT
  			GGAAGCCCTGCAGGGAGTCCA
  			ACTTCCACGGAAGAAGGAACG
  			TCTACAGAGCCATCAGAGGGG
  			TCCGCACCAGGTACCAGCGAAT
  			CCGCTACTCCCGAATCTGGCCC
  			TGGGTCCGAACCTGCCACCTCC
  			GGCTCTGAAACTCCAGGGACCT
  			CCGAATCTGCCACACCCGAGA
  			GCGGCCCTGGCTCCGAGCCCGC
  			AACATCTGGCAGCGAGACACC
  			TGGCACCTCCGAGAGCGCAAC
  			ACCCGAGAGCGGCCCTGGCAC
  			CAGCACCGAGCCATCCGAGGG
  			ATCCGCCCCAGGCACTTCTGAG
  			TCAGCCACACCCGAAAGCGGA
  			CCAGGATCACCCGCTGGCTCCC
  			CCACCAGTACCGAGGAGGGGT
  			CCCCCGCTGGAAGTCCAACAA
  			GCACTGAGGAAGGGTCCCCTG
  			CCGGCTCCCCCACAAGTACCGA
  			AGAGGGCACAAGTGAGAGCGC
  			CACTCCCGAGTCCGGGCCTGGC
  			ACCAGCACAGAGCCTTCCGAG
  			GGGTCCGCACCAGGTACCTCAG
  			AGTCTGCTACCCCCGAGTCAGG
  			GCCAGGATCAGAGCCAGCCAC
  			CTCCGGGTCTGAGACACCCGGG
  			ACTTCCGAGAGTGCCACCCCTG
  			AGTCCGGACCCGGGTCCGAGC
  			CCGCCACTTCCGGCTCCGAAAC
  			TCCCGGCACAAGCGAGAGCGC
  			TACCCCAGAGTCAGGACCAGG
  			AACATCTACAGAGCCCTCTGAA
  			GGCTCCGCTCCAGGGTCCCCAG
  			CCGGCAGTCCCACTAGCACCGA
  			GGAGGGAACCTCTGAAAGCGC
  			CACACCCGAATCAGGGCCAGG
  			GTCTGAGCCTGCTACCAGCGGC
  			AGCGAGACACCAGGCACCTCT
  			GAGTCCGCCACACCAGAGTCC
  			GGACCCGGATCTCCCGCTGGGA
  			GCCCCACCTCCACTGAGGAGG
  			GATCTCCTGCTGGCTCTCCAAC
  			ATCTACTGAGGAAGGTACCTCA
  			ACCGAGCCATCCGAGGGATCA
  			GCTCCCGGCACCTCAGAGTCGG
  			CAACCCCGGAGTCTGGACCCG
  			GAACTTCCGAAAGTGCCACACC
  			AGAGTCCGGTCCCGGGACTTCA
  			GAATCAGCAACACCCGAGTCC
  			GGCCCTGGGTCTGAACCCGCCA
  			CAAGTGGTAGTGAGACACCAG
  			GATCAGAACCTGCTACCTCAGG
  			GTCAGAGACACCCGGATCTCCG
  			GCAGGCTCACCAACCTCCACTG
  			AGGAGGGCACCAGCACAGAAC
  			CAAGCGAGGGCTCCGCACCCG
  			GAACAAGCACTGAACCCAGTG
  			AGGGTTCAGCACCCGGCTCTGA
  			GCCGGCCACAAGTGGCAGTGA
  			GACACCCGGCACTTCAGAGAG
  			TGCCACCCCCGAGAGTGGCCCA
  			GGCACTAGTACCGAGCCCTCTG
  			AAGGCAGTGCGCCAGGTTCGTC
  			TTCATAA

Example 25Expression of FVII-XTEN and FIX-XTEN
• Transient Transfection of Mammalian Cells
• Mammalian cells, including CHO-K1, BHK, COS-7, and HEK293, were found to express FVII-XTEN or FIX-XTEN using different XTEN lengths when transfected. The following are details for methods used to express the various FVII-XTEN and FIX-XTEN fusion protein constructs by transient transfection.
• HEK293 cells were plated the day before transfection, 1×10⁵per well in 12-well plate in 1 ml medium containing 10% FBS, 1× Pen/Strep, and 5 mg/ml vitamin K. For transfection the day after plating the cells, plasmid DNA (0.6 μg) diluted in OptiMEM (total 25 μl) was mixed with diluted FuGENE6 (2.1 μl FuGENE6 in 22.9 μl OptiMEM) and incubated for 30 min at room temperature before adding to the cells. Onday 3 or 4 after transfection the culture medium was collected, centrifuged at 500×g for 5 min at room temperature, and then the supernatant filtered using 0.2 μm filter before testing for expression of FVII-XTEN or FIX-XTEN in ELISA and performance in a clotting assay (PT for FVII activity and aPTT for FIX activity). The results are presented in Table 26.
• It should be noted that the titer measured for FVII-XTEN by PT assay (active FVII protein) was higher than the titer measured by ELISA (total FVII protein), and while the exact cause for this remained to be clarified, it could be due to (1) underestimation of FVII in the context of FVII-XTEN due to epitope shielding by XTEN, (2) overestimation of clotting activity by the PT assay, or a combination of both (1) and (2). It should also be noted that the titer measured for FIX by aPTT assay (active FIX protein) was significantly lower than the titer measured by ELISA (total FIX protein), only about 20%, due to unknown reasons, but one of which could be insufficient propeptide processing, a phenomenon that has been reported for recombinant FIX produced in CHO or other mammalian cells. The titer of FIX-XTEN by aPTT was even lower proportionally than ELISA compared to FIX alone, suggesting the activity of FIX could be reduced by fusing to XTEN, an assumption confirmed by analyzing the activity and ELISA titers of protein after TEV treatment for materials produced from cells transfected with plasmids constructs encoding a FVII-XTEN but with TEV cleavage site inserted in between.

• TABLE 26
  Expression of FVII, FVII-XTEN_AE864, FIX, and FIX-XTEN_AE864

  FVII

  FVII-XTEN

  FIX

  FIX-XTEN

  	ELISA	Clotting	ELISA	Clotting	ELISA	Clotting	ELISA	Clotting

  ng/ml	391.0	397.7	34.8	176.6	309.8	60.0	13.7	0.4

• Generation of CHO-K1 Stable Pools and Cell Lines that Produce FVII-XTEN
• Cells: CHO-K1 cells purchased from ATCC (Cat. CCL-61, Lot 58078551) were revived in Complete Medium (F-12K, 10% FBS and 1×P/S, Appendix 1) and passaged for four generations before multiple vials were frozen in the Complete Medium with 5% DMSO. One vial was revived in medium similar to the Complete Medium but with 5% FBS and passaged one more time before transfection.
• Generation of stable pool: Construction of plasmids pBC0014, pBC0016, and pBC0018 encoding FVII-AE864, FVII-AE864, and FVII-AE288, respectively, has been described in the Examples above. Two of the plasmids pBC0016 and pBC0018 also carry UCOE. The plasmids were first linearized with PvuII and then transfected with FuGENE6 transfection reagent into separate T25 flasks of CHO-K1 cells from above, 3.6 μg plasmid DNA for 6.5×10⁵cells per flask. Two days later the cells were transferred to T75 and cultured in Selection Medium (Complete Medium with 10 μg/ml puromycin and 5 μg/ml vitamin K). The flasks were changed to fresh Selection Medium every 2-3 days. Two weeks after transfection, cells from T75 flasks were frozen as stable pool.
• Selection of clones: For primary screening, frozen stable pool cells were revived and seeded in 6 96-well plates at a target density of 0.5 cell/well. About 1 week after seeding spent medium from wells with single cell cluster as observed under microscope were tested for expression of FVII by ELISA. The number of clones tested in the primary screening by the ELISA was: 154 for pBC0014, 210 for pBC0016, and 135 for pBC0018. Significant numbers of clones expressed no or non-detectable levels of FVII (FIG. 9, black bars, expressed as ng/ml), but 15-20% of the clones expressed FVII of 3-8 fold higher, these clones were then selected for further screening and selection, 20 for pBC0014, 30 for pBC0016, and 20 for pBC0018. The size of the cell clusters in these wells was scored 1-10 with 1 being the smallest cluster and 10 the largest cluster; the results are shown as gray bars inFIG. 9. The distribution of the cell cluster size of these clones was similar to that of all the clones for the same variant, suggesting they were selected not just because they were the fastest growers.
• For additional rounds of screening, normalized numbers of cells were seeded in multi-well plates. Spent medium were harvested 2-3 days after seeding and were tested for FVII concentration by ELISA and clotting activity by PT; Cells were also harvested from the plates and counted using Vi-Cell. Clones were ranked by (1) FVII titers according to ELISA and clotting; (2) Ratios of ELISA titer/cell count and clotting titer/cell count; and (3) Integrity and homogeneity of products produced by the clones as measured by Western blots. Selection of clones for each of the construct pBC0014, pBC0016, and pBC0018 was described separately in the following.
• pBC0014: For the second round of screening, cells in 96-well plates for the top 20 clones selected from primary screening were first expanded in T25 flasks and then seeded in duplicate 24-well plates, one cultured for 2 days and the other one for 3 days. Spent medium were collected from the plates for FVII ELISA and cells harvested and counted by Vi-Cell. Fourteen clones were selected according to titers by ELISA and clotting, ELISA titer/cell and clotting titer/cell count ratios and further screened. Frozen vials were prepared for 9 clones, 1F10, 2F7, 6H4, 1A3, 6F10, 5C2, 5F1, 3H2, 4C8. Out of the 14 clones, 1F10, 1F4, 2F7, 4C8, 6H4, and 6G1 were again screened and ranked according to titers by ELISA and clotting, ratios of ELISA titer/cell count and clotting titer/cell count, and product integrity and homogeneity by Western blot (FIGS. 10-12). Clone 6G1 expressed a product that is significantly smaller than the full-length product (FIG. 12) and was discarded. Additional frozen vials were prepared for clones 1F10, 2F7, 6H4, and 4C8. Clone 4C8 was tested for production of FVII-AE864 in roller bottles.
• pBC0016: For the second round of screening, cells in 96-well plates for the top 30 clones selected from primary screening were transferred to 12-well plates and then T25 and ranked by titers according to ELISA and clotting assays, ratios of ELISA titer/cell count and clotting titer/cell count. For the third round of screening, fifteen clones including 1D4, 1G2, 1G6, 2C11, 2H6, 3A2, 3B1, 3C7, 3F2, 3H1, 3H6, 3H10, 4G8, 5E12, 6F11 were tested, ranked according to above criteria plus Western blot (FIG. 13) and frozen cells were prepared for all of the 15 clones, except 3H6. Eight clones including 1G2, 2C11, 3B1, 3C7, 3F2, 3H10, 4G8, 5E12 were selected as the top clones and additional frozen vials were prepared for them. Clone 3H10 was selected for scale-up production in roller bottles.
• pBC0018: For the second round of screening, cells in 96-well plates for the top 20 clones selected from primary screening were first expanded in T25 flasks and then seeded in 24-well plates. Spent medium were collected from the plates for FVII ELISA and cells harvested and counted by Vi-Cell. Twelve clones were selected according to titers by ELISA and clotting, ELISA titer/cell and clotting titer/cell count ratios and further screened. Frozen vials were prepared for 9 clones, 2C3, 2D5, 3B2, 3B10, 3G2, 3G12, 5Al2, 6A3, and 6E7. Out of the 9 clones, 2D5, 3B2, 3G2, 3G12, 5Al2, 6A3, and 6E7 were again screened and ranked according to titers by ELISA and clotting, ratios of ELISA titer/cell count and clotting titer/cell count, and product integrity and homogeneity by Western blot (FIG. 14). Clone 3B2 expressed products that displayed multiple bands on Western blot and it was discarded. Additional frozen vials were prepared for clones 2D5, 3G2, 3G12, 5Al2, 6A3, 6E7. Clones 3G12 and 6E7 were used for production of FVII-AE288 in roller bottles.
• Production of FVII-XTEN Secreted in Cell Culture Medium in Roller Bottles
• CHO-K1 cell stable pools or clones were expanded in T175, 35 ml Selection Medium per flask. Cells were harvested from by trypsinization and used to seed roller bottles (1700 cm²surface area per bottle) onDay 0, 300 ml Selection Medium with cells from 1-2 T175 flasks for every roller bottle. The spent/conditioned medium was removed on Day 3 (or 4) and refilled with 300 ml fresh Selection Medium. On Day 5 (or 6) the spent/conditioned medium was removed and discarded (or harvested if XTEN fusion proteins in this medium can be purified) and 300 ml Transition Medium (UltraCHO containing 1% FBS, 0.1% Ex-Cyte, 5 mg/ml vitamin K, and 1× Pen/Step) was added to each roller bottle. On Day 7 (or 8) the spent medium was removed and discarded (or harvested if XTEN fusion proteins can be purified from this medium) and Expression Medium (OptiMEM containing 0.1% Ex-Cyte, 1% ITS-A, 5 mg/ml vitamin K, and 1× Pen/Strep) was added, 300 ml per bottle or other volumes depending on results from optimization. Conditioned medium could be harvested once everyday, or once every 2, or 3, or 4 days depending on product titer and quality desired. To harvest, the conditioned medium was poured into centrifuge bottles, and fresh Expression Medium was added, 300 ml per bottle or other volumes depending on results from optimization. This production of harvesting spent medium and refilling with fresh medium could last for 2-4 weeks until titer or/and product quality are considered too low, when the roller bottles are terminated. The conditioned medium was then centrifuged, 3500 rpm in a bench-top centrifuge, for 10 min, at 4-8° C. The supernatant was then filtered using a 0.2 mm filter. The filtrate was either processed immediately or was stored in −80° C. freezer before processing by tangential flow filtration (TFF) for purification.
Example 26Purification and Characterization of FVII-XTEN Constructs
• Concentration and Buffer Exchange of FVII-XTEN_AE864 by Tangential Flow Filtration and Diafiltration
• Supernatant batches S279, S281, S282 and S287, totaling 10.7 L in volume, from stable CHO cells lines expressing FVII-AE864 (AC404) were filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule. They were subsequently concentrated approximately 20 fold by tangential flow filtration using aMillipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge the sample was diafiltered with 10 mM tris pH 7.5, 1 mM EDTA with 5 volumes worth of buffer exchange. Samples were divided into 50 ml aliquots and frozen at −80° C. No FVII activity was detectable in the permeate fractions from the filtration and ˜75% recovery of the activity was seen in the concentrated, buffer exchanged material.
• Purification of FVII-XTEN_AE864 by BaSO₄Adsorption
• FVII-AE864 (AC404) containing supernatant was concentrated and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA. Subsequently, 5 ml of this sample was diluted 10 fold in PBS, additional NaCl was added to 50 mM, and then BaSO₄was added to 20 mg/ml. The sample was bound on a nutator at room temperature for 30 minutes. The sample was then centrifuged at 3000 rpm for 5 minutes to pellet the BaSO₄. The supernatant was discarded and the pellet resuspended in 5 ml if 200 mM sodium acetate and nutated for 30 minutes at room temperature. This was repeated two more times. After the third wash the pellet was resuspended in 0.8 ml of 100 mM trisodium citrate pH 7.0 and nutated for 30 minutes at room temperature. This was repeated once. A Bradford assay was performed to determine the total amount of protein in the sample and FVII activity was assayed in a PT based factor assay with Innovin as the activating thromboplastin (Table 27). The ratio of activity to total protein demonstrated a net Purification of ˜12 fold from this purification step.

• TABLE 27
  Purification Table of FVII-AE864 by BaSO4 Absorption

  		Total	Total
  	Volume	Activity	Protein	Specific		Purity
  Fraction	(ml)	(U)	(ug)	Activity	Purification	(%)

  Feed	5	17.3	6300	0.003	1.0	0.1%
  FT
  1	5	4.0	4687	0.001	0.3	0.0%
  Wash
  1	5	NA	57	NA	NA	NA
  Wash
  2	5	NA	8	NA	NA	NA
  Wash
  3	5	NA	8	NA	NA	NA
  Elution
  1	0.8	3.2	85	0.038	13.8	1.9%
  Elution
  2	0.8	0.4	12	0.030	11.0	1.5%

• Purification of FVII-XTEN_AE864 by aGla Affinity Chromatography
• A monoclonal antibody (clone ID CaFVII-22) which binds the GLA domain of FVII in a calcium dependent manner was coupled to Ultralink beads from Pierce. Coupling was performed by adding 10 mg of antibody in PBS to 1.25 of resin and bringing the final volume to 15 ml with coupling buffer (100 mM MOPS, 700 mM sodium citrate, pH 8.0). This produced 10 ml of resin slurry and a 1 mg to 1 ml ratio of antibody mass to bead slurry volume. The slurry was incubated for 2 hours at room temperature and then the beads were washed with coupling buffer. A BCA assay indicated ˜70% of the antibody was coupled to the beads. The beads are then quenched with 1M tris pH 8.0 for 2 hours at room temperature. The beads were equilibrated into 10 mM tris pH 7.5 and 10 mM CaCl2 and 5.5 ml of beads was mixed with 50 ml of concentrated, buffer exchanged FVII-AE864 (AC404) supernatant in 10 mM tris pH 7.5 and ˜10 mM CaCl2. The sample was incubated at 4° C. overnight on a nutator to bind the FVII-XTEN to the resin. The following day the beads were washed three times with 45ml 10 mM tris, 500 mM NaCl, 10 mM CaCl₂, pH 7.5 and then eluted with 20 ml of 10 mM tris, 100 mM EDTA, pH 7.5. SDS-PAGE analysis indicates that the purity is in excess of 90% (FIG. 15).
• Activation of FVII-XTEN_AE864 and FVII-XTEN_AE288
• Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398) were activated to FVIIa-AE864 and FVIIa-AE288 by addition of FXa. The FVII-XTEN proteins were buffer exchanged into 10 mM Tris, 10 mM CaCl2, pH7.5 via repeat rounds of concentration in an Amicon ultra 10,000 Da MWCO concentrator and subsequent dilution. The final volume was 1 ml at ˜0.4 mg/ml. FXa from Novagen was added to a final concentration of 10 units/ml and the sample incubated overnight at 4° C. Reducing SDS-PAGE indicated a complete conversion of FVII-XTEN proteins to FVIIa-XTEN proteins by the downward shift in the top band with DTT compared to the non-reduced sample which represents the loss of the light chain from the molecule, which can only occur upon activation (FIG. 16). Additionally, the light chain can be seen appearing lower on the gel and running at the same position as the light chain of control FVIIa, further confirming the transition of the FVII domain from FVII to FVIIa. Under similar buffer conditions FVII-XTEN fusions are activated to FVIIa-XTEN by the addition of thrombin, FIXa, FXIIa or any other protease capable of selectively cutting the peptide bond between R152 and I153.
• Autoactivation of FVII-XTEN_AE864 and FVII-XTEN_AE288
• Affinity purified FVII-AE864 (AC404) and FVII-AE288 (AC398) were activated to FVIIa-AE864 and FVIIa-AE288 by incubating the sample at 4° C. for 1 week. The FVII-XTEN proteins were buffer exchanged into 10 mM Tris, 10 mM CaCl₂, pH7.5 via repeat rounds of concentration in an Amicon ultra 10,000 Da MWCO concentrator and subsequent dilution. After the incubation the protein was assayed by SDS-PAGE and the top band displays the characteristic downward shift in the top band with DTT compared to the non-reduced sample which represents the loss of the light chain from the molecule, which can only occur upon activation (FIG. 17). Additionally, the light chain can bee seen appearing lower on the gel at the same point as the two lots of FXa activated material, further corroborating the conclusion that the proteins auto-activated to FVIIa-XTEN.
• Purification of FVII-XTEN_AE864 by Anion Exchange Chromatography
• A sample of FVII-AE864 (AC404) containing supernatant was concentrated and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA and then adjusted to a final concentration of ˜5 mM CaCl2 with the addition of 1M CaCl2. The sample was loaded onto a 2 ml macrocap Q column equilibrated on an Akta chromatography system. The protein was eluted with a linear gradient of 0-100% buffer B over 20 column volumes. Buffer A was comprised of 20 mM MES, 5 mM CaCl2 pH 6.0 and buffer B was comprised of 20 mM MES, 5 mM CaCl2 pH 6.0 and 500 mM NaCl. Fractions were assayed for FVII activity using a PT based factor assay with Innovin as the activating thromboplastin. A single tight peak of activity was seen eluting between 47.9 and 52.4 ml, or 23.2 to 27.8 mS/cm (FIG. 19). A Bradford assay was performed to determine the total amount of protein in the load and elution fractions. The ratio of the activity to the total protein demonstrated an ˜5 fold net purification from the column.
• Purification of FVII-XTEN_AE864 by Hydrophobic Interaction Chromatography
• A sample of FVII-AE864 (AC404) containing supernatant was concentrated and buffer exchanged into 10 mM tris pH 7.5, 1 mM EDTA and then adjusted to a final concentration of ˜5 mM CaCl2 with the addition of 1M CaCl2. The sample was loaded onto a 2 ml toyopearl phenyl column equilibrated on an Akta chromatography system. The protein was eluted with a linear gradient of 0-100% buffer B over 20 column volumes. Buffer A was comprised of 10 mM Tris, 5 mM CaCl2, 3M NaCl, pH 7.5 and buffer B was comprised of 10 mM Tris, 5 mM CaCl2, pH 7.5. Fractions were assayed for FVII activity using a PT based factor assay with Innovin as the activating thromboplastin. A single peak of activity was seen eluting between 1M and 2M NaCl (FIG. 20). A Bradford assay was performed to determine the total amount of protein in the load and elution fractions. The ratio of the activity to the total protein demonstrated an ˜2 fold net purification from the column.
• Removal of Aggregated Protein from Monomeric FVII-AE864 with Anion Exchange Chromatography
• Affinity purified FVII-AE864 (AC404) was loaded was adjusted to pH 6.0 by addition of 200 mM MES, 210 mM CaCl2 pH 6.0 at a ratio of 1 ml buffer to 10 ml sample. Using an Akta FPLC system the sample was purified using a 2 ml macrocap Q column. The column was equilibrated into buffer A (20 mM MES, 1 mM CaCl2, pH 6.0) and the sample loaded. The sample was eluted using a linear gradient of 30% to 80% buffer B (20 mM MES, 1 mM CaCl2, pH 6.0+500 mM NaCl) over 20 column volumes. The 215 nm chromatogram showed two peaks in the elution profile (FIG. 21A). The fractions corresponding to the early peak and the late peak were pooled and analyzed via size exclusion chromatography (SEC) with 60 cm BioSep G4000 column. The early peak contained a monodispersed population with a characteristic hydrodynamic radius of a monomeric AE864 protein (10.1 nm or apparent MW of 1.9 MDa) (FIG. 21B). The late peak contained two populations, the smaller monomeric peak demonstrating the absence of aggregates in the early peak. and an earlier eluting, larger peak at the void volume of the column (22 ml) characteristic of aggregated protein.
• SEC Analysis of FVII-AE864 and FVII-AE288
• FVII-AE864 (AC404) and FVII-AE288 (AC398) were purified by affinity and anion exchange chromatography and characterized. Size exclusion chromatography with 60 cm BioSep G4000 column indicated a monodispersed population with a characteristic hydrodynamic radius for either a monomeric AE864 protein (10.1 nm or apparent MW of 1.9 MDa for an apparent molecular weight factor of 15.2) or a monomeric AE288 protein (8.2 nm or apparent MW of 650 kDa for an apparent molecular weight factor of 9.0) (FIG. 18). Minimal aggregation was seen in either sample. SDS-PAGE showed a >90% pure protein with minimal host cell protein contamination.
• Lipidated Tissue Factor Initiated Clotting Activity Analysis of FVII-AE864 and FVII-AE288
• Activity was assayed by a PT based factor VII assay as follows: A standard curve was prepared by diluting normal plasma ten fold with FVII deficient plasma and then conducting 4, 5 fold serial dilutions again with factor VII deficient plasma. This created a standard curve with points at 100, 20, 4, 0.8 and 0.16 mUnits/ml of activity, where one unit of activity is defined as the amount of FVII activity in 1 ml of normal human plasma. A FVII-deficient plasma was also included to determine the background level of activity in the null plasma. The sample was prepared by adding FVII-XTEN to FVII deficient plasma at a ratio of 1:10 by volume. The samples were incubated at 37C in a molecular devices plate reader spectrophotometer for 3 minutes at which point the clotting reaction was initiated by the addition of 2 volumes of thromboplastin (Dade Innovin, B4212-50) per one volume of sample. The turbidity was monitored at 405 nm for 5 minutes to create reaction profiles. The PT time, or time to onset of clotting activity, was defined as the first time where OD405 nm increased by 0.06 over baseline. A log-linear standard curve was created with the log of activity relating linearly to the PT time. From this the activity of the sample in the plate well was determined and then the activity in the sample determined by multiplying by 11 to account for the dilution into the FVII deficient plasma. Based upon quadruplicate measurements the activity of the FVII-AE864 (AC404) fusion was 30 Units/ml and the FVII-AE288 (AC398) was 15 U/ml. Additionally, this lapidated tissue factor activation of clotting is used to assay the activity of FVII-XTEN fusions in clotting assays with more sophisticated readouts like thrombin generation assays, TEG assays, rotem assays and other in vitro/ex vivo which involve the detection of clotting enzyme function by substrate turnover, mechanical clot formation or photo-optical clot detection.
• Soluble Tissue Factor Initiated Clotting Activity Analysis of FVII-AE864 and FVII-AE288
• After activation of FVII-AE288 (AC398) to FVIIa-AE288 the activity was measured by soluble tissue factor (sTF) induced clotting. This is performed using the Stago STA-Clot FVIIa activity assays kit. Briefly, the samples were incubated with sTF, which binds and enhances FVIIa activity, but does not convert FVII to FVIIa, The time to induce a clot in FVII null plasma was defined as the first time where OD405 nm increased by 0.06 over baseline when monitored in the molecular devices plate reader. This time is compared to a standard curve comprised of known FVIIa amounts added into FVII null plasma, and an activity number calculated. The FVIIa-AE288 sample contained an activity equivalent to 112 U/ml of FVIIa activity. Additionally, this soluble tissue factor activation of clotting is used to assay the activity of FVII-XTEN fusions in clotting assays with more sophisticated readouts like thrombin generation assays, TEG assays, rotem assays and other in vitro/ex vivo which involve the detection of clotting enzyme function by substrate turnover, mechanical clot formation or photo-optical clot detection.
• ELISA Based Concentration Determination of FVII-AE864 and FVII-AE288
• FVII-XTEN fusion concentrations were determined using and ELISA assay with an affinity purified polyclonal sheep anti-human FVII antibody, where an unmodified form of the antibody is used to capture the protein and the HRP conjugated form was used to detect the protein. The capture antibody was coated at 4C overnight on to a high binding 96 well assay plate (Corning 3690). The plate was blocked with 3% BSA in PBS for 1 hour at room temperature. The plate was washed 6 times in PBST with a plate washer. Samples or standards, diluted in PBST, were then bound into the appropriate wells for 2 hours at room temperature. The standard curve ranged from 10 ng/ml to <1 pg/ml and was prepared by serially diluting commercial plasma derived FVII of a known concentration (Abcam Cat# ab62386) in PBST. The plate was again washed 6 times with PBST using a plate washer. The FVII-XTEN was then detected using the detection antibody which was bound for 1 hour at 37C. The plate was again washed 6 times with PBST using a plate washer and washed one further time with water. Signal was then developed with TMB substrate and quantified by reading at 405 nm on a molecular devices plate reader spectrophotometer. A four parameter fit is then performed on the standards and the concentration of the samples determined by comparison to the standard curve.
• Assessment of FVII-AE864 and FVII-AE288 Activity via Direct Turnover of a Fluorogenic Substrate
• FVII-XTEN fusion activity is determined by monitoring the cleavage of a peptide bond in the substrate D-FPR-6-amino-1-naphthalenesulfonamide (D-FPR-ANSN) where the DFPR moiety is a peptide chain linked to a secondary amine in the ANSH moiety. When the bond between the arginine residue and the ANSH moiety is cleaved by the serine protease activity of the FVII catalytic domain the ANSH is released and becomes an intense fluorophore. FVII-XTEN activity is measured at enzyme concentrations ranging from 50 pM to 1 μM with substrate concentrations ranging from 50 μM to 100 μM in 20 mM tris pH 8.0, 135 mM NaCl. By monitoring the change in ANSN fluorescence (excitation 352 nm, emission 470 nm) over time the activity of the FVIIa catalytic domain can be determined. This activity can be compared to a standard curve derived from FVIIa to determine the amount of FVIIa equivalents in the sample, or kinetic properties such as kcat and Km for can be determined.
• Assessment of FVII-AE864 and FVII-AE288 Activity via a FXa Coupled Chromogenic Substrate Assay
• When complexed to Tissue Factor (TF), in presence of phospholipids and Calcium, FVII and FVII-XTEN activate factor X to factor Xa. Biophen Factor VII is a chromogenic assay for testing factor VII activity. Factor VII forms an enzymatic complex with Tissue Factor, provided by rabbit Thromboplastin. It then activates factor X, present in the assay at a constant concentration and in excess, to factor Xa. The concentration of FXa is exactly measured by its activity on a specific factor Xa chromogenic substrate (SXa-11). Factor Xa cleaves the substrate and generates pNA. The amount of pNA generated is directly proportional to the factor Xa activity. Finally, there is a direct relationship between the amount of factor VII activity in the assayed sample and the factor Xa activity generated, measured by the amount of pNA released, determined by color development at 405 nm. By comparing the signal from an unknown sample to the signal from a standard curve of know FVII activity, it is possible to calculate the amount of FVII activity in an unknown sample.
Example 27ELISA Assays for FIX-XTEN Concentration Determination
• FIX-XTEN concentrations were determined using and ELISA assay with a specific matched pair of antibodies, where the detection antibody was conjugated to HRP to simplify detection (Affinity Biologicals cat# FIX-EIA). The capture antibody was coated at 4C overnight on to a high binding 96 well assay plate (Corning 3690). The plate was blocked with 3% BSA in PBS for 1 hour at room temperature. The plate was washed 6 times in PBST with a plate washer. Samples or standards, diluted in PBST, were then bound into the appropriate wells for 2 hours at room temperature. The standard curve ranged from 25 ng/ml to <1 pg/ml and was prepared by serially diluting commercial plasma derived FIX of a known concentration (Abcam Cat# ab62544) in PBST. The plate was again washed 6 times with PBST using a plate washer. The FIX was then detected using the detection antibody which was bound for 1 hour at 37C. The plate was again washed 6 times with PBST using a plate washer and washed one further time with water. Signal was then developed with TMB substrate and quantified by reading at 405 nm on a molecular devices plate reader spectrophotometer. A four parameter fit is then performed on the standards and the concentration of the samples determined by comparison to the standard curve.
Example 28aPTT Based Assays for FIX-XTEN Activity Determination
• FIX-XTEN would act to replace FIX in the intrinsic or contact activated coagulation pathway. The activity of this coagulation pathway is assessed using an activated partial thromboplastin time assay (aPTT). FIX activity specifically was measured as follows, a standard curve was prepared by diluting normal control plasma (Pacific Hemostasis cat# 100595) two fold with FIX deficient plasma (cat# 100900) and then conducting 6, 4 fold serial dilutions again with factor IX deficient plasma. This created a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 mUnits/ml of activity, where one unit of activity is defined as the amount of FIX activity in 1 ml of normal human plasma. A FIX-deficient plasma was also included to determine the background level of activity in the null plasma. The sample was prepared by adding FIX-XTEN to FIX deficient plasma at a ratio of 1:10 by volume. The samples were tested using an aPTT assay as follows. The samples were incubated at 37C in a molecular devices plate reader spectrophotometer for 2 minutes at which point an equal volume of aPTT reagent (Pacific Hemostasis cat# 100402) was added and an additional 3 minute 37C incubation performed. After the incubation the assay was activated by adding one volume of calcium chloride (Pacific Hemostasis cat# 100304). The turbidity was monitored at 450 nm for 5 minutes to create reaction profiles. The aPTT time, or time to onset of clotting activity, was defined as the first time where OD405 nm increased by 0.06 over baseline. A log-linear standard curve was created with the log of activity relating linearly to the aPTT time. From this the activity of the sample in the plate well was determined and then the activity in the sample determined by multiplying by 11 to account for the dilution into the FIX deficient plasma.
Example 29FIX/cFXI/XTEN has Enhanced Activity Compared to FIX-XTEN
• FIX (pCW0596), FIX-XTEN (pCW0597), FIX/cFXI1/XTEN (pCW0735), FIX/cFXI2/XTEN (pCW0736) and FIX/cFXI3/XTEN (pCW0737) were transiently expressed in CHO-K1 cells. Transient transfection supernatants were concentrated in 30,000 MWCO concentrators by approximately 15-fold. The concentration of the concentrated and unconcentrated samples was determined by ELISA. The clotting activity of the concentrated samples was then determined using an aPTT based factor assay. For the XTEN containing the activity was drastically altered by the presence any of the FXIc cleavage sites. In all three cases the presence of a FXI cleavage site enhanced the clotting activity by greater than 30-fold (seeFIG. 22 and Table 28). The relatively consistent ELISA measurement indicates that this is an enhancement of the specific activity, rather than a change in titer. Additionally, the ratio of the activity measurement to the ELISA concentration for the FXI cleavage site constructs was now similar to the ratios for FIX, indicating that the FIX-FXIc-XTEN contained a FIX domain of similar properties to the FIX domain expressed in the absence of XTEN.

• TABLE 28
  Activity of FIX/cFXI/XTEN Constructs

  	Concentration by	Concentration by	Fraction
  Construct	ELISA (ng/ml)	Activity (ng/ml)	Active

  FIX/cTEV/XTEN	NA	NA		4%
  FIX	197	30	15%
  FIX-XTEN	10	0	2%
  FIX/cFXI1/XTEN	23	7	30%
  FIX/cFXI2/XTEN	26	9	34%
  FIX/cFXI3/XTEN	29	11	40%

Example 30Pharmacokinetic Analysis of CFXTEN Fusion Polypeptide in Rats-FVII-XTEN_AE864
• The pharmacokinetics of the CFXTEN FVII-XTEN_AE864, compared to FVII alone, were tested in Sprague-Dawley. FVII-XTEN_AE864 and FVII were administered to female Sprague-Dawley rats (n=3) IV through a jugular vein catheter at 3 μg/rat. Blood samples (0.2 mL) were collected into prechilled heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and processed into plasma. Quantitation of the test articles was performed by ELISA assay using an anti-FVII antibody for both capture and detection. A non-compartmental analysis was performed in WinNonLin with all time points included in the fit to determine the PK parameters.
• The pharmacokinetic results are summarized in Table 29 andFIG. 23. The data show XTEN can greatly extend the half-life of FVII as a CFXTEN fusion protein as compared to FVII alone; FVII-XTEN has a half life of approximately 38 hours as compared to 1 hour for FVII. In addition FVII-XTEN was confined to the bloodstream, with a calculated volume of distribution of 50.8 mL in rats, indicating little extravasation into the extracellular space.

• TABLE 29
  Half-life of FVII test articles in rats

  	Test Article (Construct)	T½ (hrs)

  	FVII-XTEN(AP315)	37.9
  	FVII (P318)	1.0

Example 31Pharmacokinetic Analysis of CF XTEN Fusion Polypeptide in Rats-FIX-XTEN_AE864
• The pharmacokinetics of macrocap Q purified FIX-XTEN_AE864 were tested in Sprague-Dawley rats (n=3) and compared to unpurified FIX-XTEN, FIX-XTEN TEV cleaved (a preparation in which the XTEN is removed from the fusion protein by use of TEV protease), and commercially-available FIX Benefix. Compounds were administered to female Sprague-Dawley rats IV through jugular vein catheter at 3 μg/rat. Blood samples (0.2 mL) were collected into prechilled heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and processed into plasma. Quantitation of the test articles was performed by ELISA assay using an anti-FIX antibody for both capture and detection. A non-compartmental analysis was performed in WinNonLin with all time points included in the fit to determine the PK parameters.
• The pharmacokinetic results are summarized in Table 30 andFIG. 24. The data show XTEN can greatly extend the half life of FIX as a CFXTEN fusion protein as compared to either FIX-XTEN TEV cleaved or FIX Benefix; FIX-XTEN has a half life of 34.7 hours as compared to 4.6 hour for FIX Benefix. In addition FIX-XTEN was confined to the bloodstream with a calculated volume of distribution of 38 mL in rats, indicating little extravasation into the extracellular space.

• TABLE 30
  Half-life of FIX test articles in rats

  	Test Article (Construct)	T½ (hrs)

  	FIX-XTEN macro cap Q	34.7
  	(AP316a)
  	FIX-XTEN (AP316)	33.1
  	FIX-XTEN TEV (AP316b)	1.5
  	FIX Benefix	3.3

Example 32Pharmacodynamic Evaluation of FVIIa-XTEN_AE864 in animal Models
• The in vivo pharmacologic activity of FVIIa-XTEN constructs is assessed using a variety of preclinical models of bleeding including but not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models can be developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIIa approaches. FVIIa-XTEN compositions are provided in an aqueous buffer compatible with in vivo administration (for example: phosphate-buffered saline or Tris-buffered saline). The compositions are administered at appropriate doses, dosing frequency, dosing schedule and route of administration as optimized for the particular model. Efficacy determinations include measurement of FVIIa activity, prothrombin time (PT), activated partial prothrombin time (aPTT), bleeding time, whole blood clotting time (WBCT), thrombelastography (TEG or ROTEM), among others.
• In one example of a PD model, FVIIa-XTEN and FVII are administered to genetically-deficient or experimentally-induced HemA or HemB mice. At various time points post-administration, levels of FVIIa and FVIIa-XTEN are measured by ELISA, activity of FVIIa and FVIIa-XTEN are measured by commercially-available FVIIa activity kits and clotting time is measured by PT assay. Overall, the results can indicate that the FVIIa-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIIa and/or equivalent in potency to comparable dosage of FVIIa with less frequent or more convenient dosing intervals.
• In a mouse bleeding challenge PD model FVIIa-XTEN and FVIIa are administered to genetically-deficient or experimentally-induced HemA or HemB mice and effect on hemostatic challenge is measured. Hemostatic challenge can include tail transaction challenge, hemarthropthy challenge, joint bleeding or saphenous vein challenge among others. At various time points post-administration levels of FVII and FVIIa-XTEN are measured by ELISA, activity of FVII and FVIIa-XTEN are measured by commercially available FVIIa activity kit, bleeding time is measured and clotting time is measured by PT assay. Overall the results can indicate that the VIIa-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIIa and/or equivalent in potency to comparable dosage of FVIIa with less frequent or more convenient dosing intervals.
• In a dog PD model, FVIIa-XTEN and FVII are administered to genetically-deficient hemophiliac dogs. At various time points post administration, levels of FVIIa and FVIIa-XTEN are measured by ELISA, activity of FVIIa and FVIIa-XTEN are measured by commercially available FVIIa activity kit and clotting time is measured by PT assay. Overall the results can indicate that the FVIIa-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIIa and/or equivalent in potency to comparable dosage of FVIIa with less frequent or more convenient dosing.
• In a dog bleeding challenge PD model FVIIa-XTEN and FVIIa are administered to genetically deficient hemophiliac dogs and effect on hemostatic challenge is measured. Hemostatic challenge can include cuticle bleeding time among others. At various time points post administration levels of FVII and FVIIa-XTEN are measured by ELISA, activity of FVII and FVIIa-XTEN are measured by commercially available FVIIa activity kit, bleeding time is measured and clotting time is measured by PT assay. Overall the results can indicate that the VIIa-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIIa and/or equivalent in potency to comparable dosage of FVIIa with less frequent or more convenient dosing intervals.
• Additional preclinical models of bleeding include but are not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models can developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIIa approaches. Overall the results can indicate that the FVIIa-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIIa and/or equivalent in potency to comparable dosage of FVIIa with less frequent or more convenient dosing intervals.
Example 33Pharmacodynamic Evaluation of FIX-XTEN_AE864 in Animal Models
• The in vivo pharmacologic activity of FIX-XTEN constructs is assessed using a variety of preclinical models of bleeding including, but not limited to, those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models can be developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FIX approaches. FIX-XTEN compositions are provided in an aqueous buffer compatible with in vivo administration (for example: phosphate-buffered saline or Tris-buffered saline). The compositions are administered at appropriate doses, dosing frequency, dosing schedule and route of administration as optimized for the particular model. Efficacy readouts include measurement of FIX activity, PT, aPTT, bleeding time, whole blood clotting time (WBCT), thrombelastography (TEG or ROTEM), among others.
• In one example of a PD model, FIX-XTEN and FIX are administered to genetically-deficient or experimentally-induced HemA or HemB mice. At various time points post-administration, levels of FIX and FIX-XTEN are measured by ELISA, activity of FIX and FIX-XTEN are measured by commercially available FIX activity kit and clotting time is measured by aPTT assay. Overall the results can indicate that the FIX-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FIX and/or equivalent in potency to comparable dosage of FIX with less frequent or more convenient dosing intervals.
• In a mouse bleeding challenge PD model FIX-XTEN and FIX are administered to genetically deficient or experimentally induced HemA or Hem B mice and effect on hemostatic challenge is measured. Hemostatic challenge can include tail transaction challenge, hemarthropthy challenge, joint bleeding or saphenous vein challenge among others. At various time points post administration levels of FIX and FIX-XTEN are measured by ELISA, activity of FIX and FIX-XTEN are measured by commercially available FIX activity kit, bleeding time is measured and clotting time is measured by aPTT assay. Overall the results can indicate that the FIX-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FIX and/or equivalent in potency to comparable dosage of FIX with less frequent or more convenient dosing intervals.
• In a dog PD model, FIX-XTEN and FIX are administered to genetically-deficient hemophiliac dogs. At various time points post-administration, levels of FIX and FIX-XTEN are measured by ELISA, activity of FIX and FIX-XTEN are measured by commercially available FIX activity kit and clotting time is measured by aPTT assay. Overall, the results can indicate that the FIX-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FIX and/or equivalent in potency to comparable dosage of FIX with less frequent or more convenient dosing intervals.
• In a dog bleeding challenge PD model FIX a-XTEN and FIX are administered to genetically-deficient hemophiliac dogs and effect on hemostatic challenge is measured. Hemostatic challenge can include cuticle bleeding time, amongst other assays. At various time points post-administration levels of FIX and FIX-XTEN are measured by ELISA, activity of FIX and FIX-XTEN are measured by commercially available FIX activity kit, bleeding time is measured and clotting time is measured by aPTT assay. Overall, the results can indicate that the FIX-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FIX and/or equivalent in potency to comparable dosage of FIX with less frequent or more convenient dosing intervals.
• Additional preclinical models of bleeding include, but are not limited to, those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models can be developed in multiple species, including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FIX approaches. Overall the results can indicate that the FIX-XTEN constructs may be more efficacious at inhibiting bleeding as compared to FIX and/or equivalent in potency to comparable dosage of FIX with less frequent or more convenient dosing intervals.
Example 34CFXTEN with Cleavage Sequences
• C-Terminal XTEN Releasable by FXIa
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site cleavage sequence can be incorporated into the FIX-XTEN that contains an amino acid sequence that is recognized and cleaved by the FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically the amino acid sequence KLTRAET (SEQ ID NO: 6) is cut after the arginine of the sequence by FXIa protease. FXI is the pro-coagulant protease located immediately before FIX in the intrinsic or contact activated coagulation pathway. Active FXIa is produced from FXI by proteolytic cleavage of the zymogen by FXIIa. Once activated, its natural role in coagulation is to activate FIX by excising a peptide from zymogen by cutting the protein at positions R191 and R226 of FIX, which then perpetuates the coagulation pathway. Production of FXIa is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the KLTRAET cleavage sequence (SEQ ID NO: 6), the XTEN domain would only be removed from FIX concurrent with activation of the intrinsic coagulation pathway and when coagulation is required physiologically. This creates a situation where the FIX-XTEN fusion protein is processed in one additional manner during the activation of the intrinsic pathway. In addition to the natural cleavages that occur at R191 and R226 of the FIX domain by FXIa, a third cleavage would occur at the XTEN release site which would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by FXIIa
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the XTEN release site sequence can contain an amino acid sequence that is recognized and cleaved by the FXIIa protease (EC 3.4.21.38, Uniprot P00748). Specifically the sequence TMTRIVGG (SEQ ID NO: 89) is cut after the arginine atposition 4 of the sequence. FXII is a pro-coagulant protease located before FIX in the intrinsic or contact activated coagulation pathway. Active FXIIa is produced from FXII by contact with non-self surfaces and by cleavage by kallikrein. Once activated its natural role in coagulation is to activate FXI (FIG. 3) by proteolytic cleavage of the zymogen, which then in turn, perpetuates the coagulation pathway. Production of FXIIa is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the TMTRIVGG cleavage sequence (SEQ ID NO: 89), the XTEN domain would only be removed from FIX concurrent with activation of the intrinsic coagulation pathway and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of the intrinsic pathway. In addition to the natural cleavages that occur at R191 and 8226 of the FIX domain by FXIa, a third cleavage would occur at the XTEN release site that would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by Kallikrein
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the XTEN release site sequence can an amino acid sequence that is recognized and cleaved by the kallikrein protease (EC 3.4.21.34, Uniprot P03952). Specifically the sequence SPFRVVGG (SEQ ID NO: 620) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after the arginine atposition 4 of the sequence. Kallikrein is a pro-coagulant protease located before FIX in the intrinsic or contact activated coagulation pathway. Active Kallikrein is produced from Plasma Kallirien by contact with non-self surfaces. Once activated its natural role in coagulation is to activate FXII (FIG. 3) by proteolytic cleavage of the zymogen, which then in turn, perpetuates the coagulation pathway. Production of kallikrien is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the SPFRVVGG cleavage sequence (SEQ ID NO: 620) the XTEN domain would only be removed from FIX concurrent with activation of the intrinsic coagulation pathway and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of the intrinsic pathway. In addition to the natural cleavages that occur at R191 and R226 of the FIX domain by FXIa, a third cleavage would occur at the XTEN release site that would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by FVIIa
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site sequence contains an amino acid sequence that is recognized and cleaved by the FVIIa protease (EC 3.4.21.21, Uniprot P08709). Specifically the sequence LQVRIVGG (SEQ ID NO: 91) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after the arginine atposition 4 in the sequence. FVIIa is a pro-coagulant protease located before FIX in the extrinsic or cellular injury activated coagulation pathway. Active FVIIa is produced from FVII in an autocatalytic process aided by binding to tissue factor, phospholipids and calcium. Once activated its natural role in coagulation is to activate FIX and FX (FIG. 3) by proteolytic cleavage of the zymogens, which then in turn, perpetuate the coagulation pathway. FVIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the LQVRIVGG cleavage sequence (SEQ ID NO: 91) the XTEN domain would only be removed from FIX concurrent with activation of the intrinsic coagulation pathway and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of the intrinsic pathway. In addition to the natural cleavages that would occur at R191 and R226 of the FIX domain by FVIIa, a third cleavage would occur at the XTEN release site which would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by FIXa
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site cleavage sequence contains an amino acid sequence that is recognized and cleaved by the FIXa protease (EC 3.4.21.22, Uniprot P00740). Specifically the sequence PLGRIVGG (SEQ ID NO: 92) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after the arginine atposition 4 of the sequence. Active FIXa is produced by cleavage of FIX by either FXIa or FVIIa in the presence of phospholipids and calcium. Once activated its natural role in coagulation is to activate FX (FIG. 3) by proteolytic cleavage of the zymogen, which then in turn, perpetuates the coagulation pathway. FIXa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the PLGRIVGG sequence (SEQ ID NO: 92), the XTEN domain would only be removed from FIX concurrent with activation of either the extrinsic or intrinsic coagulation pathways, and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of the intrinsic pathway. In addition to the natural cleavages that would occur at R191 and R226 of the FIX domain by FVIIa or FXIa, a third cleavage would occur at the XTEN release site which would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by FXa
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the FXa protease (EC 3.4.21.6, Uniprot P00742). Specifically the sequence IEGRTVGG (SEQ ID NO: 93) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after the arginine atposition 4 in the sequence. Active FXa is produced by cleavage of FX by FIXa in the presence of phospholipids and calcium and is the step immediately down stream from factor IX in the coagulation pathway. Once activated its natural role in coagulation is to activate FII (FIG. 3) by proteolytic cleavage of the zymogen, which then in turn, perpetuates the coagulation pathway. FXa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the IEGRTVGG sequence (SEQ ID NO: 93), the XTEN domain would only be removed from FIX concurrent with activation of either the extrinsic or intrinsic coagulation pathways, and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of clotting. In addition to the natural cleavages that would occur at R191 and R226 of the FIX domain by FVIIa or FXIa, a third cleavage would occur at the XTEN release site which would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by FIIa (Thrombin)
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequence LTPRSLLV (SEQ ID NO: 94) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after the arginine atposition 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor IX in the coagulation pathway. Once activated its natural role in coagulation is to cleave fibrinogin (FIG. 3), which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the LTPRSLLV sequence (SEQ ID NO: 94), the XTEN domain would only be removed from FIX concurrent with activation of either the extrinsic or intrinsic coagulation pathways, and when coagulation is required physiologically. This creates a situation where FIX-XTEN fusion is processed in one additional manner during the activation of coagulation. In addition to the natural cleavages that would occur at R191 and R226 of the FIX domain by FVIIa or FXIa, a third cleavage would occur at the XTEN release site which would decouple the now activated FIXa from the XTEN protein. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce free FIXa which reconstitutes or augments clotting function in a subject in need thereof.
• C-Terminal XTEN Releasable by Elastase-2
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the elastase-2 protease (EC 3.4.21.37, Uniprot P08246). Specifically the sequence LGPVSGVP (SEQ ID NO: 95) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut afterposition 4 in the sequence. Elastase is constitutively expressed by neutrophils and is present at all times in the circulation. Its activity is tightly controlled by serpins and is therefore minimally active most of the time. Therefore as the long lived FIX-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FIX to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FIX.
• C-Terminal XTEN Releasable by MMP-12
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900). Specifically the sequence GPAGLGGA (SEQ ID NO: 97) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut afterposition 4 of the sequence. MMP-12 is constitutively expressed in whole blood. Therefore as the long lived FIX-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FIX to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FIX.
• C-Terminal XTEN Releasable by MMP-13
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-13 protease (EC 3.4.24.-, Uniprot P45452). Specifically the sequence GPAGLRGA (SEQ ID NO: 99) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut afterposition 4. MMP-13 is constitutively expressed in whole blood. Therefore as the long lived FIX-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FIX to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FIX.
• C-Terminal XTEN Releasable by MMP-17
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot Q9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO: 101) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut afterposition 4 in the sequence. MMP-17 is constitutively expressed in whole blood. Therefore as the long lived FIX-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FIX to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FIX.
• C-Terminal XTEN Releasable by MMP-20
• An FIX-XTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FIX can be created with a XTEN release site cleavage sequence placed in between the FIX and XTEN components, as depicted inFIG. 2F. Exemplary sequences are provided in Table 42. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot O60882). Specifically the sequence PALPLVAQ (SEQ ID NO: 102) [Rawlings N. D., et al. (2008)Nucleic Acids Res.,36: D320], is cut after position 4 (depicted by the arrow). MMP-20 is constitutively expressed in whole blood. Therefore as the long lived FIX-XTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FIX to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FIX.
• Optimization of the Release Rate of C-Terminal XTEN
• Variants of the foregoing Examples can be created in which the release rate of C-terminal XTEN is altered. As the rate of XTEN release by an XTEN release protease is dependent on the sequence of the XTEN release site, by varying the amino acid sequence in the XTEN release site one can control the rate of XTEN release. The sequence specificity of many proteases is well known in the art, and is documented in several data bases. In this case, the amino acid specificity of proteases is mapped using combinatorial libraries of substrates [Harris, J. L., et al. (2000)Proc Natl Acad Sci USA,97: 7754] or by following the cleavage of substrate mixtures as illustrated in [Schellenberger, V., et al. (1993)Biochemistry,32: 4344]. An alternative is the identification of optimal protease cleavage sequences by phage display [Matthews, D., et al. (1993)Science,260: 1113]. Constructs is made with variant sequences and assayed for XTEN release using standard assays for detection of the XTEN polypeptides.
Example 35Integration of XTEN Internal to the CF Sequence
• Internal XTEN Fusion into the KNSADK Loop (SEQ ID NO: 621)
• An FIX-XTEN fusion protein consisting of an XTEN protein inserted into a loop of FIX can be created, as depicted inFIG. 2F. Specifically, the XTEN sequence is inserted as a fusion into the KNSADNK loop (SEQ ID NO: 622) of the EGF2 domain (residues 146-152), which has no known hemophilia B mutations and is not highly structured in the FIX crystal structure. In this case, the insertion is made by dividing the native sequence at the SA bond of the loop sequence and fusing the XTEN sequence into the gap. This would give rise to a loop sequence KNS-XTEN-ADNK (‘ADNK’ disclosed as SEQ ID NO: 623). In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce FIXa-XTEN, which reconstitutes or augments clotting function in a subject in need thereof.
• Internal XTEN Fusion into the LAEN Loop (SEQ ID NO: 624)
• An FIX-XTEN fusion protein consisting of an XTEN protein inserted into a loop of FIX can be created, as depicted inFIG. 2F. Specifically, the XTEN sequence is inserted as a fusion into the LAEN loop (SEQ ID NO: 624) of the EGF2 domain (residues 163-166), which has no known hemophilia B mutations and is not highly structured in the FIX crystal structure. In this case, the insertion is made by dividing the native sequence at the AE bond of the sequence and fusing the XTEN sequence into the gap. This would give rise to a loop sequence LA-XTEN-EN. In a desirable feature of the inventive composition, this creates a situation where FIX-XTEN would remain intact as a pro-drug until activation of coagulation, at which time the molecule is processed to produce FIXa-XTEN, which reconstitutes or augments clotting function in a subject in need thereof.
• Internal XTEN Fusion into the Activation Peptide
• An FIX-XTEN fusion protein consisting of an XTEN protein inserted into a loop of FIX can be created, as depicted inFIG. 2D. Specifically, the XTEN fusion is placed into the activation peptide (residues 192-226) such that neither of the two native FXIa cleavage sites is disrupted. The insertion is made by dividing the native sequence at the T209-1210 bond of the sequence and fusing the XTEN sequence into the gap. This gives rise to a sequence starting at residue 188 of KLTRAETVFPDVDYVNSTEAET-XTEN-ILDNITQSTQSFNDFTRVVGGE (SEQ ID NOS 625 and 626, respectively). FXI is the pro-coagulant protease located immediately before FIX in the intrinsic or contact activated coagulation pathway. Active FXIa is produced from FXI by proteolytic cleavage of the zymogen by FXIIa. Once activated its natural role in coagulation is to activate FIX (FIG. 4) by excising the activation peptide from the FIX zymogen by cutting the protein at positions R191 and R226. These cuts sites are depicted by arrows and the sequences are designed to leave the P4-P4′ sites unaltered to allow for natural cleavage activity during the coagulation cascade. Therefore the XTEN domain would only be removed from FIX as part of the normal activation process within the intrinsic coagulation pathway.
• Internal XTEN Fusion in Between the FIX EGF Domains
• An FIX-XTEN fusion protein consisting of an XTEN protein inserted into a loop of FIX can be created, as depicted inFIG. 2C. Specifically, the XTEN fusion is placed in between the two EGF like domains of FIX (junction is between residues 129 and 130). The insertion is made by dividing the native sequence at the E129-L130 bond and fusing the XTEN sequence into the gap. This would give rise to a sequence starting at residue 121 of FGFEGKNCE-XTEN-LDVTCNIKNGR (SEQ ID NOS 627 and 628, respectively). Practically, this creates a situation where FIX-XTEN would circulate intact until activation of coagulation, at which time the molecule is processed to produce FIXa-XTEN, which reconstitutes or augments clotting function in an individual.
Example 36Human Clinical Trial Designs for Evaluating CFXTEN comprising FVIIa
• NovoSeven® is recombinant human coagulation factor VIIa (rFVIIa), intended for promoting hemostasis by activating the extrinsic pathway of the coagulation cascade. Due to its short half-life, NovoSeven is dosed intravenously from every 2 to every 6 hours until hemostasis is achieved. Fusion of XTEN to FVII improves the half-life of the protein, thus enabling a reduced dosing frequency using such FVII-containing fusion protein compositions.
• Clinical trials can be designed such that the efficacy and advantages of FVIIa-XTEN, relative to NovoSeven, can be verified in humans. For example, the FVIIa-XTEN, can be used in clinical trials for treatment of bleeding as performed for NovoSeven. Such studies would comprise three phases. First, a Phase I safety and pharmacokinetics study in adult patients is conducted to determine the maximum tolerated dose and pharmacokinetics and pharmacodynamics in humans (either normal subjects or patients with hemophilia), as well as to define potential toxicities and adverse events to be tracked in future studies. The study is conducted in which single rising doses of FVIIa-XTEN compositions is administered and biochemical, PK, and clinical parameters is measured. This would permit the determination of the maximum tolerated dose and establish the threshold and maximum concentrations in dosage and circulating drug that constitute the therapeutic window for the respective components. Thereafter, clinical trials is conducted in patients with the disease, disorder or condition.
• Clinical trials could be conducted in patients suffering from any disease in which NovoSeven may be expected to provide clinical benefit. For example, such indications include bleeding episodes in hemophilia A or B, patients with inhibitors to factor VIII or factor IX, and in patients with acquired hemophilia, prevention of bleeding in surgical interventions or invasive procedures in hemophilia A or B patients with inhibitors to factor VIII or factor IX and in patients with acquired hemophilia, treatment of bleeding episodes in patients with congenital FVII deficiency, and prevention of bleeding in surgical interventions or invasive procedures in patients with congenital FVII deficiency. FVIIa-XTEN may also be indicated for use in additional patient populations. Parameters and clinical endpoints are measured as a function of the dosing of the fusion proteins compositions, yielding dose-ranging information on doses that is appropriate for a subsequent Phase III trial, in addition to collecting safety data related to adverse events. The PK parameters are correlated to the physiologic, clinical and safety parameter data to establish the therapeutic window and the therapeutic dose regimen for the FVII-XTEN composition, permitting the clinician to establish the appropriate dose ranges for the composition. Finally, a phase III efficacy study is conducted wherein patients is administered the FVII-XTEN composition at the dose regimen, and a positive control (such as a commercially-available NovoSeven), or a placebo is administered using a dosing schedule deemed appropriate given the pharmacokinetic and pharmacodynamic properties of the respective compositions, with all agents administered for an appropriately extended period of time to achieve the study endpoints. Parameters that are monitored include PT assay, bleeding time assay, control of bleeding episodes, or the occurrence of spontaneous bleeding episodes; parameters that is tracked relative to the placebo or positive control groups. Efficacy outcomes is determined using standard statistical methods. Toxicity and adverse event markers are also be followed in this study to verify that the compound is safe when used in the manner described.
Example 37Human Clinical Trial Designs for Evaluating CFXTEN Comprising FIX
• BeneFIX®, Coagulation Factor IX (Recombinant), is indicated for the control and prevention of hemorrhagic episodes in patients with hemophilia B (congenital factor IX deficiency or Christmas disease), including control and prevention of bleeding in surgical settings. Dosage and duration of treatment for all factor IX products depend on the severity of the factor IX deficiency, the location and extent of bleeding, and the patient's clinical condition, age and recovery of factor IX. Fusion of XTEN to FIX improves the half-life of the protein, thus enabling a reduced dosing frequency.
• Clinical trials can be designed such that the efficacy and advantages of FIX-XTEN, relative to other factor IX clinical products, can be verified in humans. For example, the FIX-XTEN, can be used in clinical trials for treatment of hemorrhagic episodes as performed for Benefix. Such studies would comprise three phases. First, a Phase I safety and pharmacokinetics study in adult patients is conducted to determine the maximum tolerated dose and pharmacokinetics and pharmacodynamics in humans (either normal subjects or patients with hemophilia), as well as to define potential toxicities and adverse events to be tracked in future studies. The study is conducted in which single rising doses of FIX-XTEN compositions is administered and biochemical, PK, and clinical parameters is measured. This would permit the determination of the maximum tolerated dose and establish the threshold and maximum concentrations in dosage and circulating drug that constitute the therapeutic window for the respective components. Thereafter, clinical trials is conducted in patients with the disease, disorder or condition.
• Clinical trials could be conducted in patients suffering from any disease in which factor IX may be expected to provide clinical benefit. For example, such indications include the control and prevention of hemorrhagic episodes in patients with hemophilia B (congenital factor IX deficiency or Christmas disease), including control and prevention of bleeding in surgical settings. FIX-XTEN may also be indicated for use in additional patient populations. Parameters and clinical endpoints are measured as a function of the dosing of the fusion proteins compositions, yielding dose-ranging information on doses that is appropriate for a subsequent Phase III trial, in addition to collecting safety data related to adverse events. The PK parameters are correlated to the physiologic, clinical and safety parameter data to establish the therapeutic window and the therapeutic dose regimen for the FIX-XTEN composition, permitting the clinician to establish the appropriate dose ranges for the composition. Finally, a phase III efficacy study is conducted wherein patients is administered the FIX-XTEN composition at the dose regimen, and a positive control (such as a commercially-available BeneFIX), or a placebo is administered using a dosing schedule deemed appropriate given the pharmacokinetic and pharmacodynamic properties of the respective compositions, with all agents administered for an appropriately extended period of time to achieve the study endpoints. Parameters that are monitored include aPTT assay, bleeding time assay, control of bleeding episodes, or the occurrence of spontaneous bleeding episodes; parameters that is tracked relative to the placebo or positive control groups. Efficacy outcomes is determined using standard statistical methods. Toxicity and adverse event markers are also be followed in this study to verify that the compound is safe when used in the manner described.
Example 38Analytical Size Exclusion Chromatography of XTEN Fusion Proteins with Diverse Payloads
• Size exclusion chromatography analyses were performed on fusion proteins containing various therapeutic proteins and unstructured recombinant proteins of increasing length. An exemplary assay used a TSKGel-G4000 SWXL (7 8 mm×30 cm) column in which 40 μg of purified glucagon fusion protein at a concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profiles were monitored using OD214 nm and OD280 nm. Column calibration for all assays were performed using a size exclusion calibration standard from BioRad; the markers include thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographic profiles of Glucagon-Y288, Glucagon-Y144, Glucagon-Y72, Glucagon-Y36 are shown as an overlay inFIG. 35. The data show that the apparent molecular weight of each compound is proportional to the length of the attached XTEN sequence. However, the data also show that the apparent molecular weight of each construct is significantly larger than that expected for a globular protein (as shown by comparison to the standard proteins run in the same assay). Based on the SEC analyses for all constructs evaluated, including a CFXTEN composition, the apparent molecular weights, the apparent molecular weight factor (expressed as the ratio of apparent molecular weight to the calculated molecular weight) and the hydrodynamic radius (R_Hin nm) are shown in Table 31. The results indicate that incorporation of different XTENs of 576 amino acids or greater confers an apparent molecular weight for the fusion protein of approximately 339 kDa to 760, and that XTEN of 864 amino acids or greater confers an apparent molecular weight greater than approximately 800 kDA. The results of proportional increases in apparent molecular weight to actual molecular weight were consistent for fusion proteins created with XTEN from several different motif families; i.e., AD, AE, AF, AG, and AM, with increases of at least four-fold and ratios as high as about 17-fold. Additionally, the incorporation of XTEN fusion partners with 576 amino acids or more into fusion proteins with the various payloads (and 288 residues in the case of glucagon fused to Y288) resulted with a hydrodynamic radius of 7 nm or greater; well beyond the glomerular pore size of approximately 3-5 nm. Accordingly, it is expected that fusion proteins comprising growth and XTEN have reduced renal clearance, contributing to increased terminal half-life and improving the therapeutic or biologic effect relative to a corresponding un-fused biologic payload protein.

• TABLE 31
  SEC analysis of various polypeptides

  					Apparent
  Con-	XTEN or	Thera-	Actual	Apparent	Molecular
  struct	fusion	peutic	MW	MW	Weight	RH
  Name	partner	Protein	(kDa)	(kDa)	Factor	(nm)

  AC14	Y288	Glucagon	28.7	370	12.9	7.0
  AC28	Y144	Glucagon	16.1	117	7.3	5.0
  AC34	Y72	Glucagon	9.9	58.6	5.9	3.8
  AC33	Y36	Glucagon	6.8	29.4	4.3	2.6
  AC89	AF120	Glucagon	14.1	76.4	5.4	4.3
  AC88	AF108	Glucagon	13.1	61.2	4.7	3.9
  AC73	AF144	Glucagon	16.3	95.2	5.8	4.7
  AC53	AG576	GFP	74.9	339	4.5	7.0
  AC39	AD576	GFP	76.4	546	7.1	7.7
  AC41	AE576	GFP	80.4	760	9.5	8.3
  AC52	AF576	GFP	78.3	526	6.7	7.6
  AC398	AE288	FVII	76.3	650	8.5	8.2
  AC404	AE864	FVII	129	1900	14.7	10.1
  AC85	AE864	Exendin-4	83.6	938	11.2	8.9
  AC114	AM875	Exendin-4	82.4	1344	16.3	9.4
  AC143	AM875	CF	100.6	846	8.4	8.7
  AC227	AM875	IL-1ra	95.4	1103	11.6	9.2
  AC228	AM1318	IL-1ra	134.8	2286	17.0	10.5

Example 39Pharmacokinetics of Extended Polypeptides Fused to GFP in Cynomolgus Monkeys
• The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys to determine the effect of composition and length of the unstructured polypeptides on PK parameters. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are summarized inFIG. 25. They show a surprising increase of half-life with increasing length of the XTEN sequence. For example, a half-life of 10 h was determined for GFP-XTEN_L288 (with 288 amino acid residues in the XTEN). Doubling the length of the unstructured polypeptide fusion partner to 576 amino acids increased the half-life to 20-22 h for multiple fusion protein constructs; i.e., GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the unstructured polypeptide fusion partner length to 836 residues resulted in a half-life of 72-75 h for XTEN_AD836-GFP. Thus, increasing the polymer length by 288 residues from 288 to 576 residues increased in vivo half-life by about 10 h. However, increasing the polypeptide length by 260 residues from 576 residues to 836 residues increased half-life by more than 50 h. These results show that there is a surprising threshold of unstructured polypeptide length that results in a greater than proportional gain in in vivo half-life. Thus, fusion proteins comprising extended, unstructured polypeptides are expected to have the property of enhanced pharmacokinetics compared to polypeptides of shorter lengths.
Example 40Serum Stability of XTEN
• A fusion protein containing XTEN_AE864 fused to the N-terminus of GFP was incubated in monkey plasma and rat kidney lysate for up to 7 days at 37° C. Samples were withdrawn attime 0,Day 1 andDay 7 and analyzed by SDS PAGE followed by detection using Western analysis and detection with antibodies against GFP as shown inFIG. 26. The sequence of XTEN_AE864 showed negligible signs of degradation over 7 days in plasma. However, XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days. The in vivo stability of the fusion protein was tested in plasma samples wherein the GFP_AE864 was immunoprecipitated and analyzed by SDS PAGE as described above. Samples that were withdrawn up to 7 days after injection showed very few signs of degradation. The results demonstrate the resistance of CFXTEN to degradation due to serum proteases; a factor in the enhancement of pharmacokinetic properties of the CFXTEN fusion proteins.
Example 41Increasing Solubility and Stability of a Peptide Payload by Linking to XTEN
• In order to evaluate the ability of XTEN to enhance the physical/chemical properties of solubility and stability, fusion proteins of glucagon plus shorter-length XTEN were prepared and evaluated. The test articles were prepared in Tris-buffered saline at neutral pH and characterization of the Gcg-XTEN solution was by reverse-phase HPLC and size exclusion chromatography to affirm that the protein was homogeneous and non-aggregated in solution. The data are presented in Table 32. For comparative purposes, the solubility limit of unmodified glucagon in the same buffer was measured at 60 μM (0.2 mg/mL), and the result demonstrate that for all lengths of XTEN added, a substantial increase in solubility was attained. Importantly, in most cases the glucagon-XTEN fusion proteins were prepared to achieve target concentrations and were not evaluated to determine the maximum solubility limits for the given construct. However, in the case of glucagon linked to the AF-144 XTEN, the limit of solubility was determined, with the result that a 60-fold increase in solubility was achieved, compared to glucagon not linked to XTEN. In addition, the glucagon-AF144 CFXTEN was evaluated for stability, and was found to be stable in liquid formulation for at least 6 months under refrigerated conditions and for approximately one month at 37° C. (data not shown).
• The data support the conclusion that the linking of short-length XTEN polypeptides to a biologically active protein such as glucagon can markedly enhance the solubility properties of the protein by the resulting fusion protein, as well as confer stability at the higher protein concentrations.

• TABLE 32
  Solubility of Glucagon-XTEN constructs

  	TestArticle	Solubility
  	Glucagon
  	60 μM
  	Glucagon-Y36	>370 μM
  	Glucagon-Y72	>293 μM
  	Glucagon-AF108	>145 μM
  	Glucagon-AF120	>160 μM
  	Glucagon-Y144	>497 μM
  	Glucagon-AE144	>467 μM
  	Glucagon-AF144	>3600 μM
  	Glucagon-Y288	>163 μM

Example 42Analysis of Sequences for Secondary Structure by Prediction Algorithms
• Amino acid sequences can be assessed for secondary structure via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974)Biochemistry,13: 222-45) and the Garnier-Osguthorpe-Robson, or “GOR” method (Garnier J, Gibrat J F, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation.
• Several representative sequences from XTEN “families” have been assessed using two algorithm tools for the Chou-Fasman and GOR methods to assess the degree of secondary structure in these sequences. The Chou-Fasman tool was provided by William R. Pearson and the University of Virginia, at the “Biosupport” interne site, URL located on the World Wide Web at .fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it existed on Jun. 19, 2009. The GOR tool was provided by Pole Informatique Lyonnais at the Network Protein Sequence Analysis interne site, URL located on the World Wide Web at .npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl as it existed on Jun. 19, 2008.
• As a first step in the analyses, a single XTEN sequence was analyzed by the two algorithms. The AE864 composition is a XTEN with 864 amino acid residues created from multiple copies of four 12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. The sequence motifs are characterized by the fact that there is limited repetitiveness within the motifs and within the overall sequence in that the sequence of any two consecutive amino acids is not repeated more than twice in any one 12 amino acid motif, and that no three contiguous amino acids of full-length the XTEN are identical. Successively longer portions of the AF 864 sequence from the N-terminus were analyzed by the Chou-Fasman and GOR algorithms (the latter requires a minimum length of 17 amino acids). The sequences were analyzed by entering the FASTA format sequences into the prediction tools and running the analysis. The results from the analyses are presented in Table 33.
• The results indicate that, by the Chou-Fasman calculations, the four motifs of the AE family (Table 1) have no alpha-helices or beta sheets. The sequence up to 288 residues was similarly found to have no alpha-helices or beta sheets. The 432 residue sequence is predicted to have a small amount of secondary structure, with only 2 amino acids contributing to an alpha-helix for an overall percentage of 0.5%. The full-length AF864 polypeptide has the same two amino acids contributing to an alpha-helix, for an overall percentage of 0.2%. Calculations for random coil formation revealed that with increasing length, the percentage of random coil formation increased. The first 24 amino acids of the sequence had 91% random coil formation, which increased with increasing length up to the 99.77% value for the full-length sequence.
• Numerous XTEN sequences of 500 amino acids or longer from the other motif families were also analyzed and revealed that the majority had greater than 95% random coil formation. The exceptions were those sequences with one or more instances of three contiguous serine residues, which resulted in predicted beta-sheet formation. However, even these sequences still had approximately 99% random coil formation.
• In contrast, a polypeptide sequence of 84 residues limited to A, S, and P amino acids was assessed by the Chou-Fasman algorithm, which predicted a high degree of predicted alpha-helices. The sequence, which had multiple repeat “AA” and “AAA” sequences, had an overall predicted percentage of alpha-helix structure of 69%. The GOR algorithm predicted 78.57% random coil formation; far less than any sequence consisting of 12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, analyzed in the present Example.
• The analysis supports the conclusion that: 1) XTEN created from multiple sequence motifs of G, S, T, E, P, and A that have limited repetitiveness as to contiguous amino acids are predicted to have very low amounts of alpha-helices and beta-sheets; 2) that increasing the length of the XTEN does not appreciably increase the probability of alpha-helix or beta-sheet formation; and 3) that progressively increasing the length of the XTEN sequence by addition of non-repetitive 12-mers consisting of the amino acids G, S, T, E, P, and A results in increased percentage of random coil formation. In contrast, polypeptides created from amino acids limited to A, S and P that have a higher degree of internal repetitiveness are predicted to have a high percentage of alpha-helices, as determined by the Chou-Fasman algorithm, as well as random coil formation. Based on the numerous sequences evaluated by these methods, it is concluded that XTEN created from sequence motifs of G, S, T, E, P, and A that have limited repetitiveness (defined as no more than two identical contiguous amino acids in any one motif) greater than about 400 amino acid residues in length are expected to have very limited secondary structure. With the exception of motifs containing three contiguous serines, it is believed that any order or combination of sequence motifs from Table 3 can be used to create an XTEN polypeptide of a length greater than about 400 residues that will result in an XTEN sequence that is substantially devoid of secondary structure. Such sequences are expected to have the characteristics described in the CFXTEN embodiments of the invention disclosed herein.

• TABLE 33
  CHOU-FASMAN and GOR prediction calculations of polypeptide sequences

  		SEQ
  SEQ		ID		Chou-Fasman	GOR
  NAME	Sequence	NO:	No. Residues	Calculation	Calculation

  	GSTSESPSGTAP	629	12	Residue totals*: H: 0 E: 0	Not
  				percent: H: 0.0 E: 0.0	Determined
  	GTS TPESGSASP	630	12	Residue totals: H: 0 E: 0	Not
  				percent: H: 0.0 E: 0.0	Determined
  	GTSPSGESSTAP	631	12	Residue totals: H: 0 E: 0	Not
  				percent: H: 0.0 E: 0.0	Determined
  	GSTSSTAESPGP	632	12	Residue totals: H: 0 E: 0	Not
  				percent: H: 0.0 E: 0.0	Determined
  	GSPAGSPTSTEEGTSESATPES	633	24	Residue totals: H: 0 E: 0	91.67%
  	GP			percent: H: 0.0 E: 0.0
  	GSPAGSPTSTEEGTSESATPES	634	36	Residue totals: H: 0 E: 0	94.44%
  	GPGTSTEPSEGSAP			percent: H: 0.0 E: 0.0
  	GSPAGSPTSTEEGTSESATPES	635	48	Residue totals: H: 0 E: 0	93.75%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.0 E: 0.0
  	STEE
  	GSPAGSPTSTEEGTSESATPES	636	60	Residue totals: H: 0 E: 0	96.67%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.0 E: 0.0
  	STEEGTSTEPSEGSAP
  	GSPAGSPTSTEEGTSESATPES	637	108	Residue totals: H: 0 E: 0	97.22%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.0 E: 0.0
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSE TPGSEPATSGSETP
  	GSPAGSPTSTEEGTSESATPES	638	216	Residue totals: H: 0 E: 0	99.07%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.0 E: 0.0
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSESATPESGP
  	GTSTEPSEGSAPGTSTEPSEGS
  	APGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSTEPSEGSAP
  	GSPAGSPTSTEEGTSESATPES	639	432	Residue totals: H: 2 E: 3	99.54%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.5 E: 0.7
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSESATPESGP
  	GTSTEPSEGSAPGTSTEPSEGS
  	APGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSTEPSEGSAPGTSE
  	SATPESGPGSEPATSGSETPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSTEPSEGSAPGSPAGSPTST
  	EEGTSTEPSEGSAP
  AE864	GSPAGSPTSTEEGTSESATPES	640	864	Residue totals: H: 2 E: 3	99.77%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.2 E: 0.3
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSESATPESGP
  	GTSTEPSEGSAPGTSTEPSEGS
  	APGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSTEPSEGSAPGTSE
  	SATPESGPGSEPATSGSETPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSTEPSEGSAPGSPAGSPTST
  	EEGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGSEPATSGSETPGTSE
  	SATPESGPGTSTEPSEGSAPGT
  	SESATPESGPGSPAGSPTSTEE
  	GSPAGSPTSTEEGSPAGSPTST
  	EEGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGSEPAT
  	SGSETPGTSESATPESGPGSEP
  	ATSGSETPGTSESATPESGPGT
  	STEPSEGSAPGSPAGSPTSTEE
  	GTSESATPESGPGSEPATSGSE
  	TPGTSESATPESGPGSPAGSPT
  	STEEGSPAGSPTSTEEGTSTEP
  	SEGSAPGTSESATPESGPGTSE
  	SATPESGPGTSESATPESGPGS
  	EPATSGSETPGSEPATSGSETP
  	GSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGSEPATSG
  	SETPGTSESATPESGPGTSTEP
  	SEGSAP
  AD 576	GSSESGSSEGGPGSGGEPSES	641	576	Residue totals: H: 7 E: 0	99.65%
  	GSSGSSESGSSEGGPGSSESGS			percent: H: 1.2 E: 0.0
  	SEGGPGSSESGSSEGGPGSSES
  	GSSEGGPGSSESGSSEGGPGE
  	SPGGSSGSESGSEGSSGPGESS
  	GSSESGSSEGGPGSSESGSSEG
  	GPGSSESGSSEGGPGSGGEPS
  	ESGSSGESPGGSSGSESGESPG
  	GSSGSESGSGGEPSESGSSGSS
  	ESGSSEGGPGSGGEPSESGSS
  	GSGGEPSESGSSGSEGSSGPG
  	ESSGESPGGSSGSESGSGGEPS
  	ESGSSGSGGEPSESGSSGSGG
  	EPSESGSSGSSESGSSEGGPGE
  	SPGGSSGSESGESPGGSSGSES
  	GESPGGSSGSESGESPGGSSGS
  	ESGESPGGSSGSESGSSESGSS
  	EGGPGSGGEPSESGSSGSEGS
  	SGPGESSGSSESGSSEGGPGSG
  	GEPSESGSSGSSESGSSEGGPG
  	SGGEPSESGSSGESPGGSSGSE
  	SGESPGGSSGSESGSSESGSSE
  	GGPGSGGEPSESGSSGSSESGS
  	SEGGPGSGGEPSESGSSGSGG
  	EPSESGSSGESPGGSSGSESGS
  	EGSSGPGESSGSSESGSSEGGP
  	GSEGSSGPGESS
  AE576	GSPAGSPTSTEEGTSESATPES	642	576	Residue totals: H: 2 E: 0	99.65%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.4 E: 0.0
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSESATPESGP
  	GTSTEPSEGSAPGTSTEPSEGS
  	APGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSTEPSEGSAPGTSE
  	SATPESGPGSEPATSGSETPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSTEPSEGSAPGSPAGSPTST
  	EEGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGSEPATSGSETPGTSE
  	SATPESGPGTSTEPSEGSAPGT
  	SESATPESGPGSPAGSPTSTEE
  	GSPAGSPTSTEEGSPAGSPTST
  	EEGTSESATPESGPGTSTEPSE
  	GSAP
  AF540	GSTSSTAESPGPGSTSSTAESP	643	540	Residue totals: H: 2 E: 0	99.65
  	GPGSTSESPSGTAPGSTSSTAE			percent: H: 0.4 E: 0.0
  	SPGPGSTSSTAESPGPGTSTPE
  	SGSASPGSTSESPSGTAPGTSP
  	SGESSTAPGSTSESPSGTAPGS
  	TSESPSGTAPGTSPSGESSTAP
  	GSTSESPSGTAPGSTSESPSGT
  	APGTSPSGESSTAPGSTSESPS
  	GTAPGSTSESPSGTAPGSTSES
  	PSGTAPGTSTPESGSASPGSTS
  	ESPSGTAPGTSTPESGSASPGS
  	TSSTAESPGPGSTSSTAESPGP
  	GTSTPESGSASPGTSTPESGSA
  	SPGSTSESPSGTAPGTSTPESG
  	SASPGTSTPESGSASPGSTSES
  	PSGTAPGSTSESPSGTAPGSTS
  	ESPSGTAPGSTSSTAESPGPGT
  	STPESGSASPGTSTPESGSASP
  	GSTSESPSGTAPGSTSESPSGT
  	APGTSTPESGSASPGSTSESPS
  	GTAPGSTSESPSGTAPGTSTPE
  	SGSASPGTSPSGESSTAPGSTS
  	STAESPGPGTSPSGESSTAPGS
  	TSSTAESPGPGTSTPESGSASP
  	GSTSESPSGTAP
  AF504	GASPGTSSTGSPGSSPSASTGT	644	504	Residue totals: H: 0 E: 0	94.44%
  	GPGSSPSASTGTGPGTPGSGT			percent: H: 0.0 E: 0.0
  	ASSSPGSSTPSGATGSPGSNPS
  	ASTGTGPGASPGTSSTGSPGT
  	PGSGTASSSPGSSTPSGATGSP
  	GTPGSGTASSSPGASPGTSST
  	GSPGASPGTSSTGSPGTPGSG
  	TASSSPGSSTPSGATGSPGASP
  	GTSSTGSPGTPGSGTASSSPGS
  	STPSGATGSPGSNPSASTGTG
  	PGSSPSASTGTGPGSSTPSGAT
  	GSPGSSTPSGATGSPGASPGTS
  	STGSPGASPGTSSTGSPGASPG
  	TSSTGSPGTPGSGTASSSPGAS
  	PGTSSTGSPGASPGTSSTGSPG
  	ASPGTSSTGSPGSSPSASTGTG
  	PGTPGSGTASSSPGASPGTSST
  	GSPGASPGTSSTGSPGASPGTS
  	STGSPGSSTPSGATGSPGSSTP
  	SGATGSPGASPGTSSTGSPGT
  	PGSGTASSSPGSSTPSGATGSP
  	GSSTPSGATGSPGSSTPSGAT
  	GSPGSSPSASTGTGPGASPGTS
  	STGSP
  AE864	GSPAGSPTSTEEGTSESATPES	645	864	Residue totals: H: 2 E: 3	99.77%
  	GPGTSTEPSEGSAPGSPAGSPT			percent: H: 0.2 E: 0.4
  	STEEGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSESATPESGP
  	GTSTEPSEGSAPGTSTEPSEGS
  	APGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSTEPSEGSAPGTSE
  	SATPESGPGSEPATSGSETPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSTEPSEGSAPGSPAGSPTST
  	EEGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGTSESA
  	TPESGPGSEPATSGSETPGTSE
  	SATPESGPGTSTEPSEGSAPGT
  	SESATPESGPGSPAGSPTSTEE
  	GSPAGSPTSTEEGSPAGSPTST
  	EEGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGSEPAT
  	SGSETPGTSESATPESGPGSEP
  	ATSGSETPGTSESATPESGPGT
  	STEPSEGSAPGSPAGSPTSTEE
  	GTSESATPESGPGSEPATSGSE
  	TPGTSESATPESGPGSPAGSPT
  	STEEGSPAGSPTSTEEGTSTEP
  	SEGSAPGTSESATPESGPGTSE
  	SATPESGPGTSESATPESGPGS
  	EPATSGSETPGSEPATSGSETP
  	GSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGSEPATSG
  	SETPGTSESATPESGPGTSTEP
  	SEGSAP
  AF864	GSTSESPSGTAPGTSPSGESST	646	875	Residue totals: H: 2 E: 0	95.20%
  	APGSTSESPSGTAPGSTSESPS			percent: H: 0.2 E: 0.0
  	GTAPGTSTPESGSASPGTSTPE
  	SGSASPGSTSESPSGTAPGSTS
  	ESPSGTAPGTSPSGESSTAPGS
  	TSESPSGTAPGTSPSGESSTAP
  	GTSPSGESSTAPGSTSSTAESP
  	GPGTSPSGESSTAPGTSPSGES
  	STAPGSTSSTAESPGPGTSTPE
  	SGSASPGTSTPESGSASPGSTS
  	ESPSGTAPGSTSESPSGTAPGT
  	STPESGSASPGSTSSTAESPGP
  	GTSTPESGSASPGSTSESPSGT
  	APGTSPSGESSTAPGSTSSTAE
  	SPGPGTSPSGESSTAPGTSTPE
  	SGSASPGSTSSTAESPGPGSTS
  	STAESPGPGSTSSTAESPGPGS
  	TSSTAESPGPGTSPSGESSTAP
  	GSTSESPSGTAPGSTSESPSGT
  	APGTSTPESGPXXXGASASGA
  	PSTXXXXSESPSGTAPGSTSES
  	PSGTAPGSTSESPSGTAPGSTS
  	ESPSGTAPGSTSESPSGTAPGS
  	TSESPSGTAPGTSTPESGSASP
  	GTSPSGESSTAPGTSPSGESST
  	APGSTSSTAESPGPGTSPSGES
  	STAPGTSTPESGSASPGSTSES
  	PSGTAPGSTSESPSGTAPGTSP
  	SGESSTAPGSTSESPSGTAPGT
  	STPESGSASPGTSTPESGSASP
  	GSTSESPSGTAPGTSTPESGSA
  	SPGSTSSTAESPGPGSTSESPS
  	GTAPGSTSESPSGTAPGTSPSG
  	ESSTAPGSTSSTAESPGPGTSP
  	SGESSTAPGTSTPESGSASPGT
  	SPSGESSTAPGTSPSGESSTAP
  	GTSPSGESSTAPGSTSSTAESP
  	GPGSTSSTAESPGPGTSPSGES
  	STAPGSSPSASTGTGPGSSTPS
  	GATGSPGSSTPSGATGSP
  AG864	GGSPGASPGTSSTGSPGSSPSA	647	868	Residue totals: H: 0 E: 0	94.70%
  	STGTGPGSSPSASTGTGPGTP			percent: H: 0.0 E: 0.0
  	GSGTASSSPGSSTPSGATGSPG
  	SNPSASTGTGPGASPGTSSTG
  	SPGTPGSGTASSSPGSSTPSGA
  	TGSPGTPGSGTASSSPGASPG
  	TSSTGSPGASPGTSSTGSPGTP
  	GSGTASSSPGSSTPSGATGSPG
  	ASPGTSSTGSPGTPGSGTASSS
  	PGSSTPSGATGSPGSNPSASTG
  	TGPGSSPSASTGTGPGSSTPSG
  	ATGSPGSSTPSGATGSPGASP
  	GTSSTGSPGASPGTSSTGSPG
  	ASPGTSSTGSPGTPGSGTASSS
  	PGASPGTSSTGSPGASPGTSST
  	GSPGASPGTSSTGSPGSSPSAS
  	TGTGPGTPGSGTASSSPGASP
  	GTSSTGSPGASPGTSSTGSPG
  	ASPGTSSTGSPGSSTPSGATGS
  	PGSSTPSGATGSPGASPGTSST
  	GSPGTPGSGTASSSPGSSTPSG
  	ATGSPGSSTPSGATGSPGSSTP
  	SGATGSPGSSPSASTGTGPGA
  	SPGTSSTGSPGASPGTSSTGSP
  	GTPGSGTASSSPGASPGTSST
  	GSPGASPGTSSTGSPGASPGTS
  	STGSPGASPGTSSTGSPGTPGS
  	GTASSSPGSSTPSGATGSPGTP
  	GSGTASSSPGSSTPSGATGSPG
  	TPGSGTASSSPGSSTPSGATGS
  	PGSSTPSGATGSPGSSPSASTG
  	TGPGSSPSASTGTGPGASPGT
  	SSTGSPGTPGSGTASSSPGSST
  	PSGATGSPGSSPSASTGTGPGS
  	SPSASTGTGPGASPGTSSTGSP
  	GASPGTSSTGSPGSSTPSGAT
  	GSPGSSPSASTGTGPGASPGTS
  	STGSPGSSPSASTGTGPGTPGS
  	GTASSSPGSSTPSGATGSPGSS
  	TPSGATGSPGASPGTSSTGSP
  AM875	GTSTEPSEGSAPGSEPATSGSE	648	875	Residue totals: H: 7 E: 3	98.63%
  	TPGSPAGSPTSTEEGSTSSTAE			percent: H: 0.8 E: 0.3
  	SPGPGTSTPESGSASPGSTSES
  	PSGTAPGSTSESPSGTAPGTST
  	PESGSASPGTSTPESGSASPGS
  	EPATSGSETPGTSESATPESGP
  	GSPAGSPTSTEEGTSTEPSEGS
  	APGTSESATPESGPGTSTEPSE
  	GSAPGTSTEPSEGSAPGSPAG
  	SPTSTEEGTSTEPSEGSAPGTS
  	TEPSEGSAPGTSESATPESGPG
  	TSESATPESGPGTSTEPSEGSA
  	PGTSTEPSEGSAPGTSESATPE
  	SGPGTSTEPSEGSAPGSEPATS
  	GSETPGSPAGSPTSTEEGSSTP
  	SGATGSPGTPGSGTASSSPGSS
  	TPSGATGSPGTSTEPSEGSAPG
  	TSTEPSEGSAPGSEPATSGSET
  	PGSPAGSPTSTEEGSPAGSPTS
  	TEEGTSTEPSEGSAPGASASG
  	APSTGGTSESATPESGPGSPA
  	GSPTSTEEGSPAGSPTSTEEGS
  	TSSTAESPGPGSTSESPSGTAP
  	GTSPSGESSTAPGTPGSGTASS
  	SPGSSTPSGATGSPGSSPSAST
  	GTGPGSEPATSGSETPGTSES
  	ATPESGPGSEPATSGSETPGST
  	SSTAESPGPGSTSSTAESPGPG
  	TSPSGESSTAPGSEPATSGSET
  	PGSEPATSGSETPGTSTEPSEG
  	SAPGSTSSTAESPGPGTSTPES
  	GSASPGSTSESPSGTAPGTSTE
  	PSEGSAPGTSTEPSEGSAPGTS
  	TEPSEGSAPGSSTPSGATGSPG
  	SSPSASTGTGPGASPGTSSTGS
  	PGSEPATSGSETPGTSESATPE
  	SGPGSPAGSPTSTEEGSSTPSG
  	ATGSPGSSPSASTGTGPGASP
  	GTSSTGSPGTSESATPESGPGT
  	STEPSEGSAPGTSTEPSEGSAP
  AM1318	GTSTEPSEGSAPGSEPATSGSE	649	1318	Residue totals: H: 7 E: 0	99.17%
  	TPGSPAGSPTSTEEGSTSSTAE			percent: H: 0.7 E: 0.0
  	SPGPGTSTPESGSASPGSTSES
  	PSGTAPGSTSESPSGTAPGTST
  	PESGSASPGTSTPESGSASPGS
  	EPATSGSETPGTSESATPESGP
  	GSPAGSPTSTEEGTSTEPSEGS
  	APGTSESATPESGPGTSTEPSE
  	GSAPGTSTEPSEGSAPGSPAG
  	SPTSTEEGTSTEPSEGSAPGTS
  	TEPSEGSAPGTSESATPESGPG
  	TSESATPESGPGTSTEPSEGSA
  	PGTSTEPSEGSAPGTSESATPE
  	SGPGTSTEPSEGSAPGSEPATS
  	GSETPGSPAGSPTSTEEGSSTP
  	SGATGSPGTPGSGTASSSPGSS
  	TPSGATGSPGTSTEPSEGSAPG
  	TSTEPSEGSAPGSEPATSGSET
  	PGSPAGSPTSTEEGSPAGSPTS
  	TEEGTSTEPSEGSAPGPEPTGP
  	APSGGSEPATSGSETPGTSESA
  	TPESGPGSPAGSPTSTEEGTSE
  	SATPESGPGSPAGSPTSTEEGS
  	PAGSPTSTEEGTSESATPESGP
  	GSPAGSPTSTEEGSPAGSPTST
  	EEGSTSSTAESPGPGSTSESPS
  	GTAPGTSPSGESSTAPGSTSES
  	PSGTAPGSTSESPSGTAPGTSP
  	SGESSTAPGTSTEPSEGSAPGT
  	SESATPESGPGTSESATPESGP
  	GSEPATSGSETPGTSESATPES
  	GPGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGTSTEP
  	SEGSAPGTSPSGESSTAPGTSP
  	SGESSTAPGTSPSGESSTAPGT
  	STEPSEGSAPGSPAGSPTSTEE
  	GTSTEPSEGSAPGSSPSASTGT
  	GPGSSTPSGATGSPGSSTPSGA
  	TGSPGSSTPSGATGSPGSSTPS
  	GATGSPGASPGTSSTGSPGAS
  	ASGAPSTGGTSPSGESSTAPG
  	STSSTAESPGPGTSPSGESSTA
  	PGTSESATPESGPGTSTEPSEG
  	SAPGTSTEPSEGSAPGSSPSAS
  	TGTGPGSSTPSGATGSPGASP
  	GTSSTGSPGTSTPESGSASPGT
  	SPSGESSTAPGTSPSGESSTAP
  	GTSESATPESGPGSEPATSGSE
  	TPGTSTEPSEGSAPGSTSESPS
  	GTAPGSTSESPSGTAPGTSTPE
  	SGSASPGSPAGSPTSTEEGTSE
  	SATPESGPGTSTEPSEGSAPGS
  	PAGSPTSTEEGTSESATPESGP
  	GSEPATSGSETPGSSTPSGATG
  	SPGASPGTSSTGSPGSSTPSGA
  	TGSPGSTSESPSGTAPGTSPSG
  	ESSTAPGSTSSTAESPGPGSST
  	PSGATGSPGASPGTSSTGSPG
  	TPGSGTASSSPGSPAGSPTSTE
  	EGSPAGSPTSTEEGTSTEPSEG
  	SAP
  AM923	MAEPAGSPTSTEEGASPGTSS	650	924	Residue totals: H: 4 E: 3	98.70%
  	TGSPGSSTPSGATGSPGSSTPS			percent: H: 0.4 E: 0.3
  	GATGSPGTSTEPSEGSAPGSEP
  	ATSGSETPGSPAGSPTSTEEGS
  	TSSTAESPGPGTSTPESGSASP
  	GSTSESPSGTAPGSTSESPSGT
  	APGTSTPESGSASPGTSTPESG
  	SASPGSEPATSGSETPGTSESA
  	TPESGPGSPAGSPTSTEEGTST
  	EPSEGSAPGTSESATPESGPGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGTSESATP
  	ESGPGTSESATPESGPGTSTEP
  	SEGSAPGTSTEPSEGSAPGTSE
  	SATPESGPGTSTEPSEGSAPGS
  	EPATSGSETPGSPAGSPTSTEE
  	GSSTPSGATGSPGTPGSGTAS
  	SSPGSSTPSGATGSPGTSTEPS
  	EGSAPGTSTEPSEGSAPGSEPA
  	TSGSETPGSPAGSPTSTEEGSP
  	AGSPTSTEEGTSTEPSEGSAPG
  	ASASGAPSTGGTSESATPESG
  	PGSPAGSPTSTEEGSPAGSPTS
  	TEEGSTSSTAESPGPGSTSESP
  	SGTAPGTSPSGESSTAPGTPGS
  	GTASSSPGSSTPSGATGSPGSS
  	PSASTGTGPGSEPATSGSETPG
  	TSESATPESGPGSEPATSGSET
  	PGSTSSTAESPGPGSTSSTAES
  	PGPGTSPSGESSTAPGSEPATS
  	GSETPGSEPATSGSETPGTSTE
  	PSEGSAPGSTSSTAESPGPGTS
  	TPESGSASPGSTSESPSGTAPG
  	TSTEPSEGSAPGTSTEPSEGSA
  	PGTSTEPSEGSAPGSSTPSGAT
  	GSPGSSPSASTGTGPGASPGTS
  	STGSPGSEPATSGSETPGTSES
  	ATPESGPGSPAGSPTSTEEGSS
  	TPSGATGSPGSSPSASTGTGP
  	GASPGTSSTGSPGTSESATPES
  	GPGTSTEPSEGSAPGTSTEPSE
  	GSAP
  AE912	MAEPAGSPTSTEEGTPGSGTA	651	913	Residue totals: H: 8 E: 3	99.45%
  	SSSPGSSTPSGATGSPGASPGT			percent: H: 0.9 E: 0.3
  	SSTGSPGSPAGSPTSTEEGTSE
  	SATPESGPGTSTEPSEGSAPGS
  	PAGSPTSTEEGTSTEPSEGSAP
  	GTSTEPSEGSAPGTSESATPES
  	GPGSEPATSGSETPGSEPATSG
  	SETPGSPAGSPTSTEEGTSESA
  	TPESGPGTSTEPSEGSAPGTST
  	EPSEGSAPGSPAGSPTSTEEGT
  	STEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSTEPSEGS
  	APGTSESATPESGPGSEPATSG
  	SETPGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSESATPESGPGTSE
  	SATPESGPGSPAGSPTSTEEGT
  	SESATPESGPGSEPATSGSETP
  	GTSESATPESGPGTSTEPSEGS
  	APGTSTEPSEGSAPGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSTEPSEGSAPGSPA
  	GSPTSTEEGTSTEPSEGSAPGT
  	SESATPESGPGSEPATSGSETP
  	GTSESATPESGPGSEPATSGSE
  	TPGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGSPAG
  	SPTSTEEGSPAGSPTSTEEGSP
  	AGSPTSTEEGTSESATPESGPG
  	TSTEPSEGSAPGTSESATPESG
  	PGSEPATSGSETPGTSESATPE
  	SGPGSEPATSGSETPGTSESAT
  	PESGPGTSTEPSEGSAPGSPAG
  	SPTSTEEGTSESATPESGPGSE
  	PATSGSETPGTSESATPESGPG
  	SPAGSPTSTEEGSPAGSPTSTE
  	EGTSTEPSEGSAPGTSESATPE
  	SGPGTSESATPESGPGTSESAT
  	PESGPGSEPATSGSETPGSEPA
  	TSGSETPGSPAGSPTSTEEGTS
  	TEPSEGSAPGTSTEPSEGSAPG
  	SEPATSGSETPGTSESATPESG
  	PGTSTEPSEGSAP
  BC 864	GTSTEPSEPGSAGTSTEPSEPG	652		Residue totals: H: 0 E: 0	99.77%
  	SAGSEPATSGTEPSGSGASEPT			percent: H: 0 E: 0
  	STEPGSEPATSGTEPSGSEPAT
  	SGTEPSGSEPATSGTEPSGSGA
  	SEPTSTEPGTSTEPSEPGSAGS
  	EPATSGTEPSGTSTEPSEPGSA
  	GSEPATSGTEPSGSEPATSGTE
  	PSGTSTEPSEPGSAGTSTEPSE
  	PGSAGSEPATSGTEPSGSEPAT
  	SGTEPSGTSEPSTSEPGAGSGA
  	SEPTSTEPGTSEPSTSEPGAGS
  	EPATSGTEPSGSEPATSGTEPS
  	GTSTEPSEPGSAGTSTEPSEPG
  	SAGSGASEPTSTEPGSEPATSG
  	TEPSGSEPATSGTEPSGSEPAT
  	SGTEPSGSEPATSGTEPSGTST
  	EPSEPGSAGSEPATSGTEPSGS
  	GASEPTSTEPGTSTEPSEPGSA
  	GSEPATSGTEPSGSGASEPTST
  	EPGTSTEPSEPGSAGSGASEPT
  	STEPGSEPATSGTEPSGSGASE
  	PTSTEPGSEPATSGTEPSGSGA
  	SEPTSTEPGTSTEPSEPGSAGS
  	EPATSGTEPSGSGASEPTSTEP
  	GTSTEPSEPGSAGSEPATSGTE
  	PSGTSTEPSEPGSAGSEPATSG
  	TEPSGTSTEPSEPGSAGTSTEP
  	SEPGSAGTSTEPSEPGSAGTST
  	EPSEPGSAGTSTEPSEPGSAGT
  	STEPSEPGSAGTSEPSTSEPGA
  	GSGASEPTSTEPGTSTEPSEPG
  	SAGTSTEPSEPGSAGTSTEPSE
  	PGSAGSEPATSGTEPSGSGAS
  	EPTSTEPGSEPATSGTEPSGSE
  	PATSGTEPSGSEPATSGTEPSG
  	SEPATSGTEPSGTSEPSTSEPG
  	AGSEPATSGTEPSGSGASEPTS
  	TEPGTSTEPSEPGSAGSEPATS
  	GTEPSGSGASEPTSTEPGTSTE
  	PSEPGSA
  	ASPAAPAPASPAAPAPSAPAA	653	84	Residue totals: H: 58 E: 0	78.57%
  	APASPAPAAPSAPAPAAPSAA			percent: H: 69.0 E: 0.0
  	SPAAPSAPPAAASPAAPSAPP
  	AASAAAPAAASAAASAPSAAA
  *H: alpha-helix E: beta-sheet

Example 43Analysis of Polypeptide Sequences for Repetitiveness
• Polypeptide amino acid sequences can be assessed for repetitiveness by quantifying the number of times a shorter subsequence appears within the overall polypeptide. For example, a polypeptide of 200 amino acid residues has 192 overlapping 9-amino acid subsequences (or 9-mer “frames”), but the number of unique 9-mer subsequences will depend on the amount of repetitiveness within the sequence. In the present analysis, different sequences were assessed for repetitiveness by summing the occurrence of all unique 3-mer subsequences for each 3-amino acid frame across the first 200 amino acids of the polymer portion divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. The resulting subsequence score is a reflection of the degree of repetitiveness within the polypeptide.
• The results, shown in Table 34, indicate that the unstructured polypeptides consisting of 2 or 3 amino acid types have high subsequence scores, while those of consisting of 12 amino acids motifs of the six amino acids G, S, T, E, P, and A with a low degree of internal repetitiveness, have subsequence scores of less than 10, and in some cases, less than 5. For example, the L288 sequence has two amino acid types and has short, highly repetitive sequences, resulting in a subsequence score of 50.0. The polypeptide J288 has three amino acid types but also has short, repetitive sequences, resulting in a subsequence score of 33.3. Y576 also has three amino acid types, but is not made of internal repeats, reflected in the subsequence score of 15.7 over the first 200 amino acids. W576 consists of four types of amino acids, but has a higher degree of internal repetitiveness, e.g., “GGSG” (SEQ ID NO: 654), resulting in a subsequence score of 23.4. The AD576 consists of four types of 12 amino acid motifs, each consisting of four types of amino acids. Because of the low degree of internal repetitiveness of the individual motifs, the overall subsequence score over the first 200 amino acids is 13.6. In contrast, XTEN's consisting of four motifs contains six types of amino acids, each with a low degree of internal repetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5).
• Conclusions: The results indicate that the combination of 12 amino acid subsequence motifs, each consisting of four to six amino acid types that are essentially non-repetitive, into a longer XTEN polypeptide results in an overall sequence that is non-repetitive. This is despite the fact that each subsequence motif may be used multiple times across the sequence. In contrast, polymers created from smaller numbers of amino acid types resulted in higher subsequence scores, although the actual sequence can be tailored to reduce the degree of repetitiveness to result in lower subsequence scores.

• TABLE 34
  Subsequence score calculations of polypeptide sequences

  Seq		SEQ ID
  Name	Amino Acid Sequence	NO:	Score

  J288	GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE	655	33.3
  	GGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGG
  	EGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSG
  	GEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGS
  	GGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGG
  	SGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEG
  	GSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGEGGSGGE
  	G
  K288	GEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEG	656	46.9
  	GGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGG
  	EGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGG
  	GEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGE
  	GEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGG
  	EGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEG
  	EGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGEGGEGEGGGE
  	G
  L288	SSESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESS	657	50.0
  	SESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSS
  	ESSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSE
  	SSESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSES
  	SESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSESSSESS
  	ESSSSESSSESSESSSSESSSESSESSSSESSSESSESSSSES
  Y288	GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEG	658	26.8
  	SEGEGGSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSE
  	GGSEGEGGSEGSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEG
  	SGEGSEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEG
  	EGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGGEGS
  	EGEGSGEGGEGEGSEGGSEGEGGSEGGEGEGSEGSGEGEGSE
  	GGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE
  Q576	GGKPGEGGKPEGGGGKPGGKPEGEGEGKPGGKPEGGGKPGG	659	18.5
  	GEGGKPEGGKPEGEGKPGGGEGKPGGKPEGGGGKPEGEGKP
  	GGGGGKPGGKPEGEGKPGGGEGGKPEGKPGEGGEGKPGGKP
  	EGGGEGKPGGGKPGEGGKPGEGKPGGGEGGKPEGGKPEGEG
  	KPGGGEGKPGGKPGEGGKPEGGGEGKPGGKPGEGGEGKPGG
  	GKPEGEGKPGGGKPGGGEGGKPEGEGKPGGKPEGGGEGKPG
  	GKPEGGGKPEGGGEGKPGGGKPGEGGKPGEGEGKPGGKPEG
  	EGKPGGEGGGKPEGKPGGGEGGKPEGGKPGEGGKPEGGKPG
  	EGGEGKPGGGKPGEGGKPEGGGKPEGEGKPGGGGKPGEGGK
  	PEGGKPEGGGEGKPGGGKPEGEGKPGGGEGKPGGKPEGGGG
  	KPGEGGKPEGGKPGGEGGGKPEGEGKPGGKPGEGGGGKPGG
  	KPEGEGKPGEGGEGKPGGKPEGGGEGKPGGKPEGGGEGKPG
  	GGKPGEGGKPEGGGKPGEGGKPGEGGKPEGEGKPGGGEGKP
  	GGKPGEGGKPEGGGEGKPGGKPGGEGGGKPEGGKPGEGGKP
  	EG
  U576	GEGKPGGKPGSGGGKPGEGGKPGSGEGKPGGKPGSGGSGKP	660	18.1
  	GGKPGEGGKPEGGSGGKPGGGGKPGGKPGGEGSGKPGGKPE
  	GGGKPEGGSGGKPGGKPEGGSGGKPGGKPGSGEGGKPGGGK
  	PGGEGKPGSGKPGGEGSGKPGGKPEGGSGGKPGGKPEGGSGG
  	KPGGSGKPGGKPGEGGKPEGGSGGKPGGSGKPGGKPEGGGS
  	GKPGGKPGEGGKPGSGEGGKPGGGKPGGEGKPGSGKPGGEG
  	SGKPGGKPGSGGEGKPGGKPEGGSGGKPGGGKPGGEGKPGS
  	GGKPGEGGKPGSGGGKPGGKPGGEGEGKPGGKPGEGGKPGG
  	EGSGKPGGGGKPGGKPGGEGGKPEGSGKPGGGSGKPGGKPE
  	GGGGKPEGSGKPGGGGKPEGSGKPGGGKPEGGSGGKPGGSG
  	KPGGKPGEGGGKPEGSGKPGGGSGKPGGKPEGGGKPEGGSG
  	GKPGGKPEGGSGGKPGGKPGGEGSGKPGGKPGSGEGGKPGG
  	KPGEGSGGKPGGKPEGGSGGKPGGSGKPGGKPEGGGSGKPG
  	GKPGEGGKPGGEGSGKPGGSGKPG
  W576	GGSGKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPG	661	23.4
  	SGKPGGGSGKPGSGKPGGGGKPGSGSGKPGGGKPGGSGGKP
  	GGGSGKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGGSGG
  	KPGKPGSGGGSGKPGKPGSGGSGGKPGKPGSGGSGGKPGKPG
  	SGGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPGSGGSGK
  	PGSGKPGSGSGKPGSGKPGGGSGKPGSGKPGSGGSGKPGKPG
  	SGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGGSGGKPGGSG
  	GKPGKPGSGGGSGKPGKPGSGGGSGKPGKPGGSGSGKPGSGK
  	PGGGSGKPGSGKPGSGGSGKPGKPGSGGSGGKPGKPGSGGGK
  	PGSGSGKPGGGKPGSGSGKPGGGKPGSGSGKPGGGKPGSGSG
  	KPGGSGKPGSGKPGGGSGGKPGKPGSGGSGKPGSGKPGSGGS
  	GKPGKPGGSGSGKPGSGKPGGGSGKPGSGKPGGGSGKPGSGK
  	PGGGSGKPGSGKPGGGGKPGSGSGKPGGSGGKPGKPGSGGSG
  	GKPGKPGSGGSGKPGSGKPGGGSGGKPGKPGSGG
  Y576	GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGS	662	15.7
  	GEGEGGEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGS
  	EGGSEGEGGSEGGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGE
  	GSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGSGEGEGSEGGSE
  	GEGGSEGSEGEGSGEGSEGEGGSEGSEGEGGGEGSEGEGSGEG
  	SEGEGGSEGSEGEGGSEGSEGEGGEGSGEGEGSEGSGEGEGSG
  	EGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSGEGSEGEG
  	SEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEGGSEGSEG
  	EGGEGSGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSG
  	EGEGGSEGSEGEGSEGSGEGEGGEGSGEGEGSGEGSEGEGGG
  	EGSEGEGSEGSGEGEGSEGSGEGEGSEGGSEGEGGSEGSEGEG
  	SEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEGSGEGSEG
  	EGGSEGGEGEGSEGGSEGEGSEGGSEGEGGEGSGEGEGGGEG
  	SEGEGSEGSGEGEGSGEGSE
  AD576	GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSE	663	13.6
  	GGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPG
  	GSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSESGSSEGGPGS
  	SESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSE
  	SGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSE
  	SGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGE
  	PSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGE
  	SPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSE
  	SGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSS
  	EGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPG
  	GSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGS
  	SESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSE
  	SGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS
  AE576	AGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT	664	6.1
  	STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA
  	TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGT
  	STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA
  	PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES
  	ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA
  	PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
  	GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA
  	TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
  	STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
  	EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
  AF540	GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESP	665	8.8
  	GPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGE
  	SSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSE
  	SPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGS
  	TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAP
  	GTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGS
  	ASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPES
  	GSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSS
  	TAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGS
  	TSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAP
  	GTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESST
  	APGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP
  AF504	GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS	666	7.0
  	SSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGTPGSG
  	TASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGAS
  	PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSP
  	GTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPSASTG
  	TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGT
  	SSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGAS
  	PGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP
  	GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
  	GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS
  	GATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGA
  	SPGTSSTGSP
  AE864	GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS	667	6.1
  	TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPAT
  	SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTS
  	TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
  	GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPE
  	SGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
  	TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE
  	PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
  	GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
  	SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT
  	SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
  	TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
  	GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
  	SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA
  	TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
  	PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE
  	GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
  	SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP
  	SEGSAP
  AF864	GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGT	668	7.5
  	APGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPS
  	GTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPS
  	GESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGS
  	TSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAP
  	GSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGS
  	ASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSG
  	ESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTS
  	STAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPG
  	STSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGT
  	APGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPS
  	GTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPS
  	GESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGS
  	TSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAP
  	GTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGS
  	ASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSG
  	ESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSP
  	SGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPG
  	STSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATG
  	SPGSSTPSGATGSP
  AG868	GGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS	669	7.5
  	GTASSSPGSSTPSGATGSPGSNPSASTGTGPGASPGTSSTGSPGT
  	PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGS
  	PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSS
  	TGSPGTPGSGTASSSPGSSTPSGATGSPGSNPSASTGTGPGSSPS
  	ASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPG
  	ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTG
  	SPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGT
  	ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSST
  	PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG
  	SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
  	GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS
  	STGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG
  	SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
  	TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT
  	GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
  	ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP
  	GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG
  	SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG
  	SPGASPGTSSTGSP
  AM875	GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAES	670	4.5
  	PGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPES
  	GSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
  	TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS
  	APGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
  	EGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPA
  	GSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
  	TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
  	EEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATP
  	ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSE
  	SPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGS
  	SPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
  	PGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSG
  	SETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPE
  	SGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
  	TEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP
  	GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGAT
  	GSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEP
  	SEGSAPGTSTEPSEGSAP
  AM1318	GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAES	671	4.5
  	PGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPES
  	GSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG
  	TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS
  	APGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPS
  	EGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPA
  	GSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
  	TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST
  	EEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSG
  	SETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAG
  	SPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSP
  	AGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAP
  	GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGS
  	APGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESAT
  	PESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTST
  	EPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPG
  	TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGT
  	GPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG
  	ATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSS
  	TAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGT
  	STEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGS
  	PGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPE
  	SGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESP
  	SGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTST
  	EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
  	SSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGT
  	APGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTS
  	STGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTST
  	EPSEGSAP

Example 44Calculation of TEPITOPE Scores
• TEPITOPE scores of 9mer peptide sequence can be calculated by adding pocket potentials as described by Sturniolo [Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example, separate Tepitope scores were calculated for individual HLA alleles. Table 35 shows as an example the pocket potentials for HLA*0101B, which occurs in high frequency in the Caucasian population. To calculate the TEPITOPE score of a peptide with sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual pocket potentials in Table 35 were added. The HLA*0101B score of a 9mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 672) is the sum of 0, −1.3, 0, 0.9, 0, −1.8, 0.09, 0, 0.
• To evaluate the TEPITOPE scores for long peptides one can repeat the process for all 9mer subsequences of the sequences. This process can be repeated for the proteins encoded by other HLA alleles. Tables 36-39 give pocket potentials for the protein products of HLA alleles that occur with high frequency in the Caucasian population.
• TEPITOPE scores calculated by this method range from approximately −10 to +10. However, 9mer peptides that lack a hydrophobic amino acid (FKLMVWY (SEQ ID NO: 673)) in P1 position have calculated TEPITOPE scores in the range of −1009 to −989. This value is biologically meaningless and reflects the fact that a hydrophobic amino acid serves as an anchor residue for HLA binding and peptides lacking a hydrophobic residue in P1 are considered non binders to HLA. Because most XTEN sequences lack hydrophobic residues, all combinations of 9mer subsequences will have TEPITOPEs in the range in the range of −1009 to −989. This method confirms that XTEN polypeptides may have few or no predicted T-cell epitopes.

• TABLE 35
  Pocket potential for HLA*0101B allele.

  Amino Acid	P1	P2	P3	P4	P5	P6	P7	P8	P9

  A	−999	0	0	0	—	0	0	—	0
  C	−999	0	0	0	—	0	0	—	0
  D	−999	−1.3	−1.3	−2.4	—	−2.7	−2	—	−1.9
  E	−999	0.1	−1.2	−0.4	—	−2.4	−0.6	—	−1.9
  F	0	0.8	0.8	0.08	—	−2.1	0.3	—	−0.4
  G	−999	0.5	0.2	−0.7	—	−0.3	−1.1	—	−0.8
  H	−999	0.8	0.2	−0.7	—	−2.2	0.1	—	−1.1
  I	−1	1.1	1.5	0.5	—	−1.9	0.6	—	0.7
  K	−999	1.1	0	−2.1	—	−2	−0.2	—	−1.7
  L	−1	1	1	0.9	—	−2	0.3	—	0.5
  M	−1	1.1	1.4	0.8	—	−1.8	0.09	—	0.08
  N	−999	0.8	0.5	0.04	—	−1.1	0.1	—	−1.2
  P	−999	−0.5	0.3	−1.9	—	−0.2	0.07	—	−1.1
  Q	−999	1.2	0	0.1	—	−1.8	0.2	—	−1.6
  R	−999	2.2	0.7	−2.1	—	−1.8	0.09	—	−1
  S	−999	−0.3	0.2	−0.7	—	−0.6	−0.2	—	−0.3
  T	−999	0	0	−1	—	−1.2	0.09	—	−0.2
  V	−1	2.1	0.5	−0.1	—	−1.1	0.7	—	0.3
  W	0	−0.1	0	−1.8	—	−2.4	−0.1	—	−1.4
  Y	0	0.9	0.8	−1.1	—	−2	0.5	—	−0.9

• TABLE 36
  Pocket potential for HLA*0301B allele.

  Amino acid	P1	P2	P3	P4	P5	P6	P7	P8	P9

  A	−999	0	0	0	—	0	0	—	0
  C	−999	0	0	0	—	0	0	—	0
  D	−999	−1.3	−1.3	2.3	—	−2.4	−0.6	—	−0.6
  E	−999	0.1	−1.2	−1	—	−1.4	−0.2	—	−0.3
  F	−1	0.8	0.8	−1	—	−1.4	0.5	—	0.9
  G	−999	0.5	0.2	0.5	—	−0.7	0.1	—	0.4
  H	−999	0.8	0.2	0	—	−0.1	−0.8	—	−0.5
  I	0	1.1	1.5	0.5	—	0.7	0.4	—	0.6
  K	−999	1.1	0	−1	—	1.3	−0.9	—	−0.2
  L	0	1	1	0	—	0.2	0.2	—	−0
  M	0	1.1	1.4	0	—	−0.9	1.1	—	1.1
  N	−999	0.8	0.5	0.2	—	−0.6	−0.1	—	−0.6
  P	−999	−0.5	0.3	−1	—	0.5	0.7	—	−0.3
  Q	−999	1.2	0	0	—	−0.3	−0.1	—	−0.2
  R	−999	2.2	0.7	−1	—	1	−0.9	—	0.5
  S	−999	−0.3	0.2	0.7	—	−0.1	0.07	—	1.1
  T	−999	0	0	−1	—	0.8	−0.1	—	−0.5
  V	0	2.1	0.5	0	—	1.2	0.2	—	0.3
  W	−1	−0.1	0	−1	—	−1.4	−0.6	—	−1
  Y	−1	0.9	0.8	−1	—	−1.4	−0.1	—	0.3

• TABLE 37
  Pocket potential for HLA*0401B allele.

  Amino acid	P1	P2	P3	P4	P5	P6	P7	P8	P9

  A	−999	0	0	0	—	0	0	—	0
  C	−999	0	0	0	—	0	0	—	0
  D	−999	−1.3	−1.3	1.4	—	−1.1	−0.3	—	−1.7
  E	−999	0.1	−1.2	1.5	—	−2.4	0.2	—	−1.7
  F	0	0.8	0.8	−0.9	—	−1.1	−1	—	−1
  G	−999	0.5	0.2	−1.6	—	−1.5	−1.3	—	−1
  H	−999	0.8	0.2	1.1	—	−1.4	0	—	0.08
  I	−1	1.1	1.5	0.8	—	−0.1	0.08	—	−0.3
  K	−999	1.1	0	−1.7	—	−2.4	−0.3	—	−0.3
  L	−1	1	1	0.8	—	−1.1	0.7	—	−1
  M	−1	1.1	1.4	0.9	—	−1.1	0.8	—	−0.4
  N	−999	0.8	0.5	0.9	—	1.3	0.6	—	−1.4
  P	−999	−0.5	0.3	−1.6	—	0	−0.7	—	−1.3
  Q	−999	1.2	0	0.8	—	−1.5	0	—	0.5
  R	−999	2.2	0.7	−1.9	—	−2.4	−1.2	—	−1
  S	−999	−0.3	0.2	0.8	—	1	−0.2	—	0.7
  T	−999	0	0	0.7	—	1.9	−0.1	—	−1.2
  V	−1	2.1	0.5	−0.9	—	0.9	0.08	—	−0.7
  W	0	−0.1	0	−1.2	—	−1	−1.4	—	−1
  Y	0	0.9	0.8	−1.6	—	−1.5	−1.2	—	−1

• TABLE 38
  Pocket potential for HLA*0701B allele.

  Amino acid	P1	P2	P3	P4	P5	P6	P7	P8	P9

  A	−999	0	0	0	—	0	0	—	0
  C	−999	0	0	0	—	0	0	—	0
  D	−999	−1.3	−1.3	−1.6	—	−2.5	−1.3	—	−1.2
  E	−999	0.1	−1.2	−1.4	—	−2.5	0.9	—	−0.3
  F	0	0.8	0.8	0.2	—	−0.8	2.1	—	2.1
  G	−999	0.5	0.2	−1.1	—	−0.6	0	—	−0.6
  H	−999	0.8	0.2	0.1	—	−0.8	0.9	—	−0.2
  I	−1	1.1	1.5	1.1	—	−0.5	2.4	—	3.4
  K	−999	1.1	0	−1.3	—	−1.1	0.5	—	−1.1
  L	−1	1	1	−0.8	—	−0.9	2.2	—	3.4
  M	−1	1.1	1.4	−0.4	—	−0.8	1.8	—	2
  N	−999	0.8	0.5	−1.1	—	−0.6	1.4	—	−0.5
  P	−999	−0.5	0.3	−1.2	—	−0.5	−0.2	—	−0.6
  Q	−999	1.2	0	−1.5	—	−1.1	1.1	—	−0.9
  R	−999	2.2	0.7	−1.1	—	−1.1	0.7	—	−0.8
  S	−999	−0.3	0.2	1.5	—	0.6	0.4	—	−0.3
  T	−999	0	0	1.4	—	−0.1	0.9	—	0.4
  V	−1	2.1	0.5	0.9	—	0.1	1.6	—	2
  W	0	−0.1	0	−1.1	—	−0.9	1.4	—	0.8
  Y	0	0.9	0.8	−0.9	—	−1	1.7	—	1.1

• TABLE 39
  Pocket potential for HLA*1501B allele.

  Amino acid	P1	P2	P3	P4	P5	P6	P7	P8	P9

  A	−999	0	0	0	—	0	0	—	0
  C	−999	0	0	0	—	0	0	—	0
  D	−999	−1.3	−1.3	−0.4	—	−0.4	−0.7	—	−1.9
  E	−999	0.1	−1.2	−0.6	—	−1	−0.7	—	−1.9
  F	−1	0.8	0.8	2.4	—	−0.3	1.4	—	−0.4
  G	−999	0.5	0.2	0	—	0.5	0	—	−0.8
  H	−999	0.8	0.2	1.1	—	−0.5	0.6	—	−1.1
  I	0	1.1	1.5	0.6	—	0.05	1.5	—	0.7
  K	−999	1.1	0	−0.7	—	−0.3	−0.3	—	−1.7
  L	0	1	1	0.5	—	0.2	1.9	—	0.5
  M	0	1.1	1.4	1	—	0.1	1.7	—	0.08
  N	−999	0.8	0.5	−0.2	—	0.7	0.7	—	−1.2
  P	−999	−0.5	0.3	−0.3	—	−0.2	0.3	—	−1.1
  Q	−999	1.2	0	−0.8	—	−0.8	−0.3	—	−1.6
  R	−999	2.2	0.7	0.2	—	1	−0.5	—	−1
  S	−999	−0.3	0.2	−0.3	—	0.6	0.3	—	−0.3
  T	−999	0	0	−0.3	—	−0	0.2	—	−0.2
  V	0	2.1	0.5	0.2	—	−0.3	0.3	—	0.3
  W	−1	−0.1	0	0.4	—	−0.4	0.6	—	−1.4
  Y	−1	0.9	0.8	2.5	—	0.4	0.7	—	−0.9

• TABLE 40
  Exemplary Biological Activity, Exemplary Assays and Preferred Indications

  Biologically Active		Exemplary Activity
  Protein	Biological Activity	Assay	Preferred Indication:
  Factor IX	Coagulation factor IX is	Factor IX clotting	Hemophilia B; bleeding;
  (Coagulation	a vitamin K-dependent	activity: Valder	Factor IX deficiency;
  factor IX	factor that circulates in	R. et al., 2001	Christmas disease;
  (human);	the blood as an inactive	“Posttranslational	bleeding episodes in
  Factor IX	zymogen. Factor IX is	modifications of	patients with factor VIII
  Complex;	converted to an active	recombinant	inhibitor or Factor
  Christmas factor;	form by factor XIa,	myotube-	VII deficiency
  plasma	which excises the	synthesized human
  thromboplastin	activation peptide and	factor IX” Blood 97:
  component	thus generates a heavy	130-138.
  (PTC);	chain and a light chain	Activated partial
  prothrombin	held together by one or	thromboplastin
  complex	more disulfide bonds. In	time: Rao LV,
  concentrate	the blood coagulation	Activation of
  (PCC);	cascade, activated	human factor VII
  Nonacog alpha;	factor IX activates factor	during clotting in
  MONONINE;	X to its active form	vitro Blood. 1985;
  ALPHANINE-	through interactions	65(1): 218-26;
  SD;	with Ca+2 ions,	Park CH, A
  BEBULIN;	membrane	diagnostic
  PROPLEX-	phospholipids, and	challenge: mild
  T; KONYNE;	factor VIII. Alterations of	hemophilia B with
  PROFILNINE	this gene, including	normal activated
  SD;	point mutations,	partial
  BeneFIX;	insertions and deletions,	thromboplastin
  IMMUNINE	cause factor IX	time. Blood Coagul
  VH)	deficiency, which is a	Fibrinolysis. 2010
  	recessive X-linked	Jun; 21(4): 368-71.
  	disorder, also
  	called hemophilia B or
  	Christmas disease.
  Factor VII	Coagulation factor VII is	Coagulation Assay	Bleeding Disorders;
  (Coagulation	a vitamin K-dependent	using Prothrombin	Coronary Restenosis;
  Factor VII;	factor essential for	Clotting Time	Hemophilia A and B; Liver
  Active-site	hemostasis. This	(Belaaouaj AA et	Disorders; Thrombosis;
  inactivated factor	factor circulates in the	al., J. Biol. Chem.	Vascular Restenosis;
  VII	blood in a zymogen	275: 27123-8,	Surgery-related
  (DEGR-	form, and is converted	2000; Diaz-Collier	hemorrhagic episodes
  VIIa/FFR-	to an active form by	JA et al., Thromb
  VIIa); Eptacog	either factor IXa, factor	Haemost 71: 339-46,
  alfa;	Xa, factor XIIa, or	1994).
  Coagulation	thrombin by minor
  Factor	proteolysis. Upon
  VIIa; Novoseven;	activation of the factor
  NiaStase;	VII, a heavy chain
  Novostase;	containing a catalytic
  MONOCLATE-	domain and a light
  P)	chain containing 2 EGF-
  	like domains are
  	generated, and two
  	chains are held together
  	by a disulfide bond.
  	In the presence of factor
  	III and calcium ions, the
  	activated factor then
  	further activates the
  	coagulation cascade by
  	converting factor IX to
  	factor IXa and/or factor
  	X to factor Xa. Defects
  	in this gene can cause
  	coagulopathy.

• TABLE 41
  Exemplary CFXTEN comprising CF and single XTEN

  CFXTEN		SEQ ID		SEQ ID
  Name*	Amino Acid Sequence	NO:	DNA Nucleotide Sequence	NO:
  FVII-	ANAFLEELRPGSLE	674	GCCAACGCGTTCCTGGAGGAGC	675
  AE288	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GTSESATPESGPGS		AGGTCATCATCCCCAGCACGTA
  	EPATSGSETPGTSE		CGTCCCGGGCACCACCAACCAC
  	SATPESGPGSEPAT		GACATCGCGCTGCTCCGCCTGC
  	SGSETPGTSESATP		ACCAGCCCGTGGTCCTCACTGA
  	ESGPGTSTEPSEGS		CCATGTGGTGCCCCTCTGCCTG
  	APGSPAGSPTSTEE		CCCGAACGGACGTTCTCTGAGA
  	GTSESATPESGPGS		GGACGCTGGCCTTCGTGCGCTT
  	EPATSGSETPGTSE		CTCATTGGTCAGCGGCTGGGGC
  	SATPESGPGSPAGS		CAGCTGCTGGACCGTGGCGCCA
  	PTSTEEGSPAGSPT		CGGCCCTGGAGCTCATGGTCCT
  	STEEGTSTEPSEGS		CAACGTGCCCCGGCTGATGACC
  	APGTSESATPESGP		CAGGACTGCCTGCAGCAGTCAC
  	GTSESATPESGPGT		GGAAGGTGGGAGACTCCCCAA
  	SESATPESGPGSEP		ATATCACGGAGTACATGTTCTG
  	ATSGSETPGSEPAT		TGCCGGCTACTCGGATGGCAGC
  	SGSETPGSPAGSPT		AAGGACTCCTGCAAGGGGGAC
  	STEEGTSTEPSEGS		AGTGGAGGCCCACATGCCACCC
  	APGTSTEPSEGSAP		ACTACCGGGGCACGTGGTACCT
  	GSEPATSGSETPGT		GACGGGCATCGTCAGCTGGGGC
  	SESATPESGPGTST		CAGGGCTGCGCAACCGTGGGCC
  	EPSEGSAP		ACTTTGGGGTGTACACCAGGGT
  			CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGTGGTACC
  			TCTGAAAGCGCAACTCCTGAGT
  			CTGGCCCAGGTAGCGAACCTGC
  			TACCTCCGGCTCTGAGACTCCA
  			GGTACCTCTGAAAGCGCAACCC
  			CGGAATCTGGTCCAGGTAGCGA
  			ACCTGCAACCTCTGGCTCTGAA
  			ACCCCAGGTACCTCTGAAAGCG
  			CTACTCCTGAATCTGGCCCAGG
  			TACTTCTACTGAACCGTCCGAG
  			GGCAGCGCACCAGGTAGCCCTG
  			CTGGCTCTCCAACCTCCACCGA
  			AGAAGGTACCTCTGAAAGCGCA
  			ACCCCTGAATCCGGCCCAGGTA
  			GCGAACCGGCAACCTCCGGTTC
  			TGAAACCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAGTCCGGCC
  			CAGGTAGCCCGGCTGGCTCTCC
  			GACTTCCACCGAGGAAGGTAGC
  			CCGGCTGGCTCTCCAACTTCTA
  			CTGAAGAAGGTACTTCTACCGA
  			ACCTTCCGAGGGCAGCGCACCA
  			GGTACTTCTGAAAGCGCTACCC
  			CTGAGTCCGGCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAATCC
  			GGTCCAGGTACTTCTGAAAGCG
  			CTACCCCGGAATCTGGCCCAGG
  			TAGCGAACCGGCTACTTCTGGT
  			TCTGAAACCCCAGGTAGCGAAC
  			CGGCTACCTCCGGTTCTGAAAC
  			TCCAGGTAGCCCAGCAGGCTCT
  			CCGACTTCCACTGAGGAAGGTA
  			CTTCTACTGAACCTTCCGAAGG
  			CAGCGCACCAGGTACCTCTACT
  			GAACCTTCTGAGGGCAGCGCTC
  			CAGGTAGCGAACCTGCAACCTC
  			TGGCTCTGAAACCCCAGGTACC
  			TCTGAAAGCGCTACTCCTGAAT
  			CTGGCCCAGGTACTTCTACTGA
  			ACCGTCCGAGGGCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	676	GCCAACGCGTTCCTGGAGGAGC	677
  AE864	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GSPAGSPTSTEEGT		AGGTCATCATCCCCAGCACGTA
  	SESATPESGPGTST		CGTCCCGGGCACCACCAACCAC
  	EPSEGSAPGSPAGS		GACATCGCGCTGCTCCGCCTGC
  	PTSTEEGTSTEPSE		ACCAGCCCGTGGTCCTCACTGA
  	GSAPGTSTEPSEGS		CCATGTGGTGCCCCTCTGCCTG
  	APGTSESATPESGP		CCCGAACGGACGTTCTCTGAGA
  	GSEPATSGSETPGS		GGACGCTGGCCTTCGTGCGCTT
  	EPATSGSETPGSPA		CTCATTGGTCAGCGGCTGGGGC
  	GSPTSTEEGTSESA		CAGCTGCTGGACCGTGGCGCCA
  	TPESGPGTSTEPSE		CGGCCCTGGAGCTCATGGTCCT
  	GSAPGTSTEPSEGS		CAACGTGCCCCGGCTGATGACC
  	APGSPAGSPTSTEE		CAGGACTGCCTGCAGCAGTCAC
  	GTSTEPSEGSAPGT		GGAAGGTGGGAGACTCCCCAA
  	STEPSEGSAPGTSE		ATATCACGGAGTACATGTTCTG
  	SATPESGPGTSTEP		TGCCGGCTACTCGGATGGCAGC
  	SEGSAPGTSESATP		AAGGACTCCTGCAAGGGGGAC
  	ESGPGSEPATSGSE		AGTGGAGGCCCACATGCCACCC
  	TPGTSTEPSEGSAP		ACTACCGGGGCACGTGGTACCT
  	GTSTEPSEGSAPGT		GACGGGCATCGTCAGCTGGGGC
  	SESATPESGPGTSE		CAGGGCTGCGCAACCGTGGGCC
  	SATPESGPGSPAGS		ACTTTGGGGTGTACACCAGGGT
  	PTSTEEGTSESATP		CTCCCAGTACATCGAGTGGCTG
  	ESGPGSEPATSGSE		CAAAAGCTCATGCGCTCAGAGC
  	TPGTSESATPESGP		CACGCCCAGGAGTCCTCCTGCG
  	GTSTEPSEGSAPGT		AGCCCCATTTCCCGGTGGTAGC
  	STEPSEGSAPGTST		CCGGCTGGCTCTCCTACCTCTAC
  	EPSEGSAPGTSTEP		TGAGGAAGGTACTTCTGAAAGC
  	SEGSAPGTSTEPSE		GCTACTCCTGAGTCTGGTCCAG
  	GSAPGTSTEPSEGS		GTACCTCTACTGAACCGTCCGA
  	APGSPAGSPTSTEE		AGGTAGCGCTCCAGGTAGCCCA
  	GTSTEPSEGSAPGT		GCAGGCTCTCCGACTTCCACTG
  	SESATPESGPGSEP		AGGAAGGTACTTCTACTGAACC
  	ATSGSETPGTSESA		TTCCGAAGGCAGCGCACCAGGT
  	TPESGPGSEPATSG		ACCTCTACTGAACCTTCTGAGG
  	SETPGTSESATPES		GCAGCGCTCCAGGTACTTCTGA
  	GPGTSTEPSEGSAP		AAGCGCTACCCCGGAATCTGGC
  	GTSESATPESGPGS		CCAGGTAGCGAACCGGCTACTT
  	PAGSPTSTEEGSPA		CTGGTTCTGAAACCCCAGGTAG
  	GSPTSTEEGSPAGS		CGAACCGGCTACCTCCGGTTCT
  	PTSTEEGTSESATP		GAAACTCCAGGTAGCCCGGCAG
  	ESGPGTSTEPSEGS		GCTCTCCGACCTCTACTGAGGA
  	APGTSESATPESGP		AGGTACTTCTGAAAGCGCAACC
  	GSEPATSGSETPGT		CCGGAGTCCGGCCCAGGTACCT
  	SESATPESGPGSEP		CTACCGAACCGTCTGAGGGCAG
  	ATSGSETPGTSESA		CGCACCAGGTACTTCTACCGAA
  	TPESGPGTSTEPSE		CCGTCCGAGGGTAGCGCACCAG
  	GSAPGSPAGSPTST		GTAGCCCAGCAGGTTCTCCTAC
  	EEGTSESATPESGP		CTCCACCGAGGAAGGTACTTCT
  	GSEPATSGSETPGT		ACCGAACCGTCCGAGGGTAGCG
  	SESATPESGPGSPA		CACCAGGTACCTCTACTGAACC
  	GSPTSTEEGSPAGS		TTCTGAGGGCAGCGCTCCAGGT
  	PTSTEEGTSTEPSE		ACTTCTGAAAGCGCTACCCCGG
  	GSAPGTSESATPES		AGTCCGGTCCAGGTACTTCTAC
  	GPGTSESATPESGP		TGAACCGTCCGAAGGTAGCGCA
  	GTSESATPESGPGS		CCAGGTACTTCTGAAAGCGCAA
  	EPATSGSETPGSEP		CCCCTGAATCCGGTCCAGGTAG
  	ATSGSETPGSPAGS		CGAACCGGCTACTTCTGGCTCT
  	PTSTEEGTSTEPSE		GAGACTCCAGGTACTTCTACCG
  	GSAPGTSTEPSEGS		AACCGTCCGAAGGTAGCGCACC
  	APGSEPATSGSETP		AGGTACTTCTACTGAACCGTCT
  	GTSESATPESGPGT		GAAGGTAGCGCACCAGGTACTT
  	STEPSEGSAP		CTGAAAGCGCAACCCCGGAATC
  			CGGCCCAGGTACCTCTGAAAGC
  			GCAACCCCGGAGTCCGGCCCAG
  			GTAGCCCTGCTGGCTCTCCAAC
  			CTCCACCGAAGAAGGTACCTCT
  			GAAAGCGCAACCCCTGAATCCG
  			GCCCAGGTAGCGAACCGGCAAC
  			CTCCGGTTCTGAAACCCCAGGT
  			ACCTCTGAAAGCGCTACTCCGG
  			AGTCTGGCCCAGGTACCTCTAC
  			TGAACCGTCTGAGGGTAGCGCT
  			CCAGGTACTTCTACTGAACCGT
  			CCGAAGGTAGCGCACCAGGTAC
  			TTCTACCGAACCGTCCGAAGGC
  			AGCGCTCCAGGTACCTCTACTG
  			AACCTTCCGAGGGCAGCGCTCC
  			AGGTACCTCTACCGAACCTTCT
  			GAAGGTAGCGCACCAGGTACTT
  			CTACCGAACCGTCCGAGGGTAG
  			CGCACCAGGTAGCCCAGCAGGT
  			TCTCCTACCTCCACCGAGGAAG
  			GTACTTCTACCGAACCGTCCGA
  			GGGTAGCGCACCAGGTACCTCT
  			GAAAGCGCAACTCCTGAGTCTG
  			GCCCAGGTAGCGAACCTGCTAC
  			CTCCGGCTCTGAGACTCCAGGT
  			ACCTCTGAAAGCGCAACCCCGG
  			AATCTGGTCCAGGTAGCGAACC
  			TGCAACCTCTGGCTCTGAAACC
  			CCAGGTACCTCTGAAAGCGCTA
  			CTCCTGAATCTGGCCCAGGTAC
  			TTCTACTGAACCGTCCGAGGGC
  			AGCGCACCAGGTACTTCTGAAA
  			GCGCTACTCCTGAGTCCGGCCC
  			AGGTAGCCCGGCTGGCTCTCCG
  			ACTTCCACCGAGGAAGGTAGCC
  			CGGCTGGCTCTCCAACTTCTACT
  			GAAGAAGGTAGCCCGGCAGGC
  			TCTCCGACCTCTACTGAGGAAG
  			GTACTTCTGAAAGCGCAACCCC
  			GGAGTCCGGCCCAGGTACCTCT
  			ACCGAACCGTCTGAGGGCAGCG
  			CACCAGGTACCTCTGAAAGCGC
  			AACTCCTGAGTCTGGCCCAGGT
  			AGCGAACCTGCTACCTCCGGCT
  			CTGAGACTCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAATCTGGT
  			CCAGGTAGCGAACCTGCAACCT
  			CTGGCTCTGAAACCCCAGGTAC
  			CTCTGAAAGCGCTACTCCTGAA
  			TCTGGCCCAGGTACTTCTACTG
  			AACCGTCCGAGGGCAGCGCACC
  			AGGTAGCCCTGCTGGCTCTCCA
  			ACCTCCACCGAAGAAGGTACCT
  			CTGAAAGCGCAACCCCTGAATC
  			CGGCCCAGGTAGCGAACCGGCA
  			ACCTCCGGTTCTGAAACCCCAG
  			GTACTTCTGAAAGCGCTACTCC
  			TGAGTCCGGCCCAGGTAGCCCG
  			GCTGGCTCTCCGACTTCCACCG
  			AGGAAGGTAGCCCGGCTGGCTC
  			TCCAACTTCTACTGAAGAAGGT
  			ACTTCTACCGAACCTTCCGAGG
  			GCAGCGCACCAGGTACTTCTGA
  			AAGCGCTACCCCTGAGTCCGGC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAATCCGGTCCAGGTAC
  			TTCTGAAAGCGCTACCCCGGAA
  			TCTGGCCCAGGTAGCGAACCGG
  			CTACTTCTGGTTCTGAAACCCC
  			AGGTAGCGAACCGGCTACCTCC
  			GGTTCTGAAACTCCAGGTAGCC
  			CAGCAGGCTCTCCGACTTCCAC
  			TGAGGAAGGTACTTCTACTGAA
  			CCTTCCGAAGGCAGCGCACCAG
  			GTACCTCTACTGAACCTTCTGA
  			GGGCAGCGCTCCAGGTAGCGAA
  			CCTGCAACCTCTGGCTCTGAAA
  			CCCCAGGTACCTCTGAAAGCGC
  			TACTCCTGAATCTGGCCCAGGT
  			ACTTCTACTGAACCGTCCGAGG
  			GCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	678	GCCAACGCGTTCCTGGAGGAGC	679
  AF864	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GSTSESPSGTAPGT		AGGTCATCATCCCCAGCACGTA
  	SPSGESSTAPGSTS		CGTCCCGGGCACCACCAACCAC
  	ESPSGTAPGSTSES		GACATCGCGCTGCTCCGCCTGC
  	PSGTAPGTSTPESG		ACCAGCCCGTGGTCCTCACTGA
  	SASPGTSTPESGSA		CCATGTGGTGCCCCTCTGCCTG
  	SPGSTSESPSGTAP		CCCGAACGGACGTTCTCTGAGA
  	GSTSESPSGTAPGT		GGACGCTGGCCTTCGTGCGCTT
  	SPSGESSTAPGSTS		CTCATTGGTCAGCGGCTGGGGC
  	ESPSGTAPGTSPSG		CAGCTGCTGGACCGTGGCGCCA
  	ESSTAPGTSPSGES		CGGCCCTGGAGCTCATGGTCCT
  	STAPGSTSSTAESP		CAACGTGCCCCGGCTGATGACC
  	GPGTSPSGESSTAP		CAGGACTGCCTGCAGCAGTCAC
  	GTSPSGESSTAPGS		GGAAGGTGGGAGACTCCCCAA
  	TSSTAESPGPGTST		ATATCACGGAGTACATGTTCTG
  	PESGSASPGTSTPE		TGCCGGCTACTCGGATGGCAGC
  	SGSASPGSTSESPS		AAGGACTCCTGCAAGGGGGAC
  	GTAPGSTSESPSGT		AGTGGAGGCCCACATGCCACCC
  	APGTSTPESGSASP		ACTACCGGGGCACGTGGTACCT
  	GSTSSTAESPGPGT		GACGGGCATCGTCAGCTGGGGC
  	STPESGSASPGSTS		CAGGGCTGCGCAACCGTGGGCC
  	ESPSGTAPGTSPSG		ACTTTGGGGTGTACACCAGGGT
  	ESSTAPGSTSSTAE		CTCCCAGTACATCGAGTGGCTG
  	SPGPGTSPSGESST		CAAAAGCTCATGCGCTCAGAGC
  	APGTSTPESGSASP		CACGCCCAGGAGTCCTCCTGCG
  	GSTSSTAESPGPGS		AGCCCCATTTCCCGGTGGTTCT
  	TSSTAESPGPGSTS		ACCAGCGAATCTCCTTCTGGCA
  	STAESPGPGSTSST		CCGCTCCAGGTACCTCTCCTAG
  	AESPGPGTSPSGES		CGGCGAATCTTCTACCGCTCCA
  	STAPGSTSESPSGT		GGTTCTACTAGCGAATCTCCTTC
  	APGSTSESPSGTAP		TGGCACTGCACCAGGTTCTACT
  	GTSTPESGPXXXG		AGCGAATCCCCGTCTGGTACTG
  	ASASGAPSTXXXX		CTCCAGGTACTTCTACTCCTGA
  	SESPSGTAPGSTSE		AAGCGGTTCCGCTTCTCCAGGT
  	SPSGTAPGSTSESP		ACCTCTACTCCGGAAAGCGGTT
  	SGTAPGSTSESPSG		CTGCATCTCCAGGTTCTACCAG
  	TAPGSTSESPSGTA		CGAATCTCCTTCTGGCACCGCT
  	PGSTSESPSGTAPG		CCAGGTTCTACTAGCGAATCCC
  	TSTPESGSASPGTS		CGTCTGGTACCGCACCAGGTAC
  	PSGESSTAPGTSPS		TTCTCCTAGCGGCGAATCTTCTA
  	GESSTAPGSTSSTA		CCGCACCAGGTTCTACTAGCGA
  	ESPGPGTSPSGESS		ATCTCCGTCTGGCACTGCTCCA
  	TAPGTSTPESGSAS		GGTACTTCTCCTAGCGGTGAAT
  	PGSTSESPSGTAPG		CTTCTACCGCTCCAGGTACTTCC
  	STSESPSGTAPGTS		CCTAGCGGCGAATCTTCTACCG
  	PSGESSTAPGSTSE		CTCCAGGTTCTACTAGCTCTACT
  	SPSGTAPGTSTPES		GCAGAATCTCCGGGCCCAGGTA
  	GSASPGTSTPESGS		CCTCTCCTAGCGGTGAATCTTCT
  	ASPGSTSESPSGTA		ACCGCTCCAGGTACTTCTCCGA
  	PGTSTPESGSASPG		GCGGTGAATCTTCTACCGCTCC
  	STSSTAESPGPGST		AGGTTCTACTAGCTCTACTGCA
  	SESPSGTAPGSTSE		GAATCTCCTGGCCCAGGTACCT
  	SPSGTAPGTSPSGE		CTACTCCGGAAAGCGGCTCTGC
  	SSTAPGSTSSTAES		ATCTCCAGGTACTTCTACCCCTG
  	PGPGTSPSGESSTA		AAAGCGGTTCTGCATCTCCAGG
  	PGTSTPESGSASPG		TTCTACTAGCGAATCTCCTTCTG
  	TSPSGESSTAPGTS		GCACTGCACCAGGTTCTACCAG
  	PSGESSTAPGTSPS		CGAATCTCCGTCTGGCACTGCA
  	GESSTAPGSTSSTA		CCAGGTACCTCTACCCCTGAAA
  	ESPGPGSTSSTAES		GCGGTTCCGCTTCTCCAGGTTCT
  	PGPGTSPSGESSTA		ACCAGCTCTACCGCAGAATCTC
  	PGSSPSASTGTGPG		CTGGTCCAGGTACCTCTACTCC
  	SSTPSGATGSPGSS		GGAAAGCGGCTCTGCATCTCCA
  	TPSGATGSP		GGTTCTACTAGCGAATCTCCTTC
  			TGGCACTGCACCAGGTACTTCT
  			CCGAGCGGTGAATCTTCTACCG
  			CACCAGGTTCTACTAGCTCTAC
  			CGCTGAATCTCCGGGCCCAGGT
  			ACTTCTCCGAGCGGTGAATCTT
  			CTACTGCTCCAGGTACCTCTACT
  			CCTGAAAGCGGTTCTGCATCTC
  			CAGGTTCCACTAGCTCTACCGC
  			AGAATCTCCGGGCCCAGGTTCT
  			ACTAGCTCTACTGCTGAATCTC
  			CTGGCCCAGGTTCTACTAGCTC
  			TACTGCTGAATCTCCGGGTCCA
  			GGTTCTACCAGCTCTACTGCTG
  			AATCTCCTGGTCCAGGTACCTC
  			CCCGAGCGGTGAATCTTCTACT
  			GCACCAGGTTCTACTAGCGAAT
  			CTCCTTCTGGCACTGCACCAGG
  			TTCTACCAGCGAATCTCCGTCT
  			GGCACTGCACCAGGTACCTCTA
  			CCCCTGAAAGCGGTCCXXXXXX
  			XXXXXXTGCAAGCGCAAGCGG
  			CGCGCCAAGCACGGGAXXXXX
  			XXXTAGCGAATCTCCTTCTGGT
  			ACCGCTCCAGGTTCTACCAGCG
  			AATCCCCGTCTGGTACTGCTCC
  			AGGTTCTACCAGCGAATCTCCT
  			TCTGGTACTGCACCAGGTTCTA
  			CTAGCGAATCTCCTTCTGGTAC
  			CGCTCCAGGTTCTACCAGCGAA
  			TCCCCGTCTGGTACTGCTCCAG
  			GTTCTACCAGCGAATCTCCTTCT
  			GGTACTGCACCAGGTACTTCTA
  			CTCCGGAAAGCGGTTCCGCATC
  			TCCAGGTACTTCTCCTAGCGGT
  			GAATCTTCTACTGCTCCAGGTA
  			CCTCTCCTAGCGGCGAATCTTCT
  			ACTGCTCCAGGTTCTACCAGCT
  			CTACTGCTGAATCTCCGGGTCC
  			AGGTACTTCCCCGAGCGGTGAA
  			TCTTCTACTGCACCAGGTACTTC
  			TACTCCGGAAAGCGGTTCCGCT
  			TCTCCAGGTTCTACCAGCGAAT
  			CTCCTTCTGGCACCGCTCCAGG
  			TTCTACTAGCGAATCCCCGTCT
  			GGTACCGCACCAGGTACTTCTC
  			CTAGCGGCGAATCTTCTACCGC
  			ACCAGGTTCTACTAGCGAATCC
  			CCGTCTGGTACCGCACCAGGTA
  			CTTCTACCCCGGAAAGCGGCTC
  			TGCTTCTCCAGGTACTTCTACCC
  			CGGAAAGCGGCTCCGCATCTCC
  			AGGTTCTACTAGCGAATCTCCT
  			TCTGGTACCGCTCCAGGTACTT
  			CTACCCCTGAAAGCGGCTCCGC
  			TTCTCCAGGTTCCACTAGCTCTA
  			CCGCTGAATCTCCGGGTCCAGG
  			TTCTACCAGCGAATCTCCTTCTG
  			GCACCGCTCCAGGTTCTACTAG
  			CGAATCCCCGTCTGGTACCGCA
  			CCAGGTACTTCTCCTAGCGGCG
  			AATCTTCTACCGCACCAGGTTC
  			TACCAGCTCTACTGCTGAATCT
  			CCGGGTCCAGGTACTTCCCCGA
  			GCGGTGAATCTTCTACTGCACC
  			AGGTACTTCTACTCCGGAAAGC
  			GGTTCCGCTTCTCCAGGTACCTC
  			CCCTAGCGGCGAATCTTCTACT
  			GCTCCAGGTACCTCTCCTAGCG
  			GCGAATCTTCTACCGCTCCAGG
  			TACCTCCCCTAGCGGTGAATCT
  			TCTACCGCACCAGGTTCTACTA
  			GCTCTACTGCTGAATCTCCGGG
  			TCCAGGTTCTACCAGCTCTACT
  			GCTGAATCTCCTGGTCCAGGTA
  			CCTCCCCGAGCGGTGAATCTTC
  			TACTGCACCAGGTTCTAGCCCT
  			TCTGCTTCCACCGGTACCGGCC
  			CAGGTAGCTCTACTCCGTCTGG
  			TGCAACTGGCTCTCCAGGTAGC
  			TCTACTCCGTCTGGTGCAACCG
  			GCTCCCCA
  FVII-	ANAFLEELRPGSLE	680	GCCAACGCGTTCCTGGAGGAGC	681
  AG864	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GASPGTSSTGSPGS		AGGTCATCATCCCCAGCACGTA
  	SPSASTGTGPGSSP		CGTCCCGGGCACCACCAACCAC
  	SASTGTGPGTPGS		GACATCGCGCTGCTCCGCCTGC
  	GTASSSPGSSTPSG		ACCAGCCCGTGGTCCTCACTGA
  	ATGSPGSNPSASTG		CCATGTGGTGCCCCTCTGCCTG
  	TGPGASPGTSSTGS		CCCGAACGGACGTTCTCTGAGA
  	PGTPGSGTASSSPG		GGACGCTGGCCTTCGTGCGCTT
  	SSTPSGATGSPGTP		CTCATTGGTCAGCGGCTGGGGC
  	GSGTASSSPGASPG		CAGCTGCTGGACCGTGGCGCCA
  	TSSTGSPGASPGTS		CGGCCCTGGAGCTCATGGTCCT
  	STGSPGTPGSGTAS		CAACGTGCCCCGGCTGATGACC
  	SSPGSSTPSGATGS		CAGGACTGCCTGCAGCAGTCAC
  	PGASPGTSSTGSPG		GGAAGGTGGGAGACTCCCCAA
  	TPGSGTASSSPGSS		ATATCACGGAGTACATGTTCTG
  	TPSGATGSPGSNPS		TGCCGGCTACTCGGATGGCAGC
  	ASTGTGPGSSPSAS		AAGGACTCCTGCAAGGGGGAC
  	TGTGPGSSTPSGAT		AGTGGAGGCCCACATGCCACCC
  	GSPGSSTPSGATGS		ACTACCGGGGCACGTGGTACCT
  	PGASPGTSSTGSPG		GACGGGCATCGTCAGCTGGGGC
  	ASPGTSSTGSPGAS		CAGGGCTGCGCAACCGTGGGCC
  	PGTSSTGSPGTPGS		ACTTTGGGGTGTACACCAGGGT
  	GTASSSPGASPGTS		CTCCCAGTACATCGAGTGGCTG
  	STGSPGASPGTSST		CAAAAGCTCATGCGCTCAGAGC
  	GSPGASPGTSSTGS		CACGCCCAGGAGTCCTCCTGCG
  	PGSSPSASTGTGPG		AGCCCCATTTCCCGGTGGTGCT
  	TPGSGTASSSPGAS		TCCCCGGGCACCAGCTCTACTG
  	PGTSSTGSPGASPG		GTTCTCCAGGTTCTAGCCCGTCT
  	TSSTGSPGASPGTS		GCTTCTACTGGTACTGGTCCAG
  	STGSPGSSTPSGAT		GTTCTAGCCCTTCTGCTTCCACT
  	GSPGSSTPSGATGS		GGTACTGGTCCAGGTACCCCGG
  	PGASPGTSSTGSPG		GTAGCGGTACCGCTTCTTCTTCT
  	TPGSGTASSSPGSS		CCAGGTAGCTCTACTCCGTCTG
  	TPSGATGSPGSSTP		GTGCTACCGGCTCTCCAGGTTC
  	SGATGSPGSSTPSG		TAACCCTTCTGCATCCACCGGT
  	ATGSPGSSPSASTG		ACCGGCCCAGGTGCTTCTCCGG
  	TGPGASPGTSSTGS		GCACCAGCTCTACTGGTTCTCC
  	PGASPGTSSTGSPG		AGGTACCCCGGGCAGCGGTACC
  	TPGSGTASSSPGAS		GCATCTTCTTCTCCAGGTAGCTC
  	PGTSSTGSPGASPG		TACTCCTTCTGGTGCAACTGGTT
  	TSSTGSPGASPGTS		CTCCAGGTACTCCTGGCAGCGG
  	STGSPGASPGTSST		TACCGCTTCTTCTTCTCCAGGTG
  	GSPGTPGSGTASSS		CTTCTCCTGGTACTAGCTCTACT
  	PGSSTPSGATGSPG		GGTTCTCCAGGTGCTTCTCCGG
  	TPGSGTASSSPGSS		GCACTAGCTCTACTGGTTCTCC
  	TPSGATGSPGTPGS		AGGTACCCCGGGTAGCGGTACT
  	GTASSSPGSSTPSG		GCTTCTTCCTCTCCAGGTAGCTC
  	ATGSPGSSTPSGAT		TACCCCTTCTGGTGCAACCGGC
  	GSPGSSPSASTGTG		TCTCCAGGTGCTTCTCCGGGCA
  	PGSSPSASTGTGPG		CCAGCTCTACCGGTTCTCCAGG
  	ASPGTSSTGSPGTP		TACCCCGGGTAGCGGTACCGCT
  	GSGTASSSPGSSTP		TCTTCTTCTCCAGGTAGCTCTAC
  	SGATGSPGSSPSAS		TCCGTCTGGTGCTACCGGCTCTC
  	TGTGPGSSPSASTG		CAGGTTCTAACCCTTCTGCATCC
  	TGPGASPGTSSTGS		ACCGGTACCGGCCCAGGTTCTA
  	PGASPGTSSTGSPG		GCCCTTCTGCTTCCACCGGTACT
  	SSTPSGATGSPGSS		GGCCCAGGTAGCTCTACCCCTT
  	PSASTGTGPGASPG		CTGGTGCTACCGGCTCCCCAGG
  	TSSTGSPGSSPSAS		TAGCTCTACTCCTTCTGGTGCAA
  	TGTGPGTPGSGTA		CTGGCTCTCCAGGTGCATCTCC
  	SSSPGSSTPSGATG		GGGCACTAGCTCTACTGGTTCT
  	SPGSSTPSGATGSP		CCAGGTGCATCCCCTGGCACTA
  	GASPGTSSTGSP		GCTCTACTGGTTCTCCAGGTGCT
  			TCTCCTGGTACCAGCTCTACTG
  			GTTCTCCAGGTACTCCTGGCAG
  			CGGTACCGCTTCTTCTTCTCCAG
  			GTGCTTCTCCTGGTACTAGCTCT
  			ACTGGTTCTCCAGGTGCTTCTCC
  			GGGCACTAGCTCTACTGGTTCT
  			CCAGGTGCTTCCCCGGGCACTA
  			GCTCTACCGGTTCTCCAGGTTCT
  			AGCCCTTCTGCATCTACTGGTA
  			CTGGCCCAGGTACTCCGGGCAG
  			CGGTACTGCTTCTTCCTCTCCAG
  			GTGCATCTCCGGGCACTAGCTC
  			TACTGGTTCTCCAGGTGCATCC
  			CCTGGCACTAGCTCTACTGGTT
  			CTCCAGGTGCTTCTCCTGGTACC
  			AGCTCTACTGGTTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCAAC
  			CGGTTCCCCAGGTAGCTCTACT
  			CCTTCTGGTGCTACTGGCTCCCC
  			AGGTGCATCCCCTGGCACCAGC
  			TCTACCGGTTCTCCAGGTACCC
  			CGGGCAGCGGTACCGCATCTTC
  			CTCTCCAGGTAGCTCTACCCCG
  			TCTGGTGCTACCGGTTCCCCAG
  			GTAGCTCTACCCCGTCTGGTGC
  			AACCGGCTCCCCAGGTAGCTCT
  			ACTCCGTCTGGTGCAACCGGCT
  			CCCCAGGTTCTAGCCCGTCTGC
  			TTCCACTGGTACTGGCCCAGGT
  			GCTTCCCCGGGCACCAGCTCTA
  			CTGGTTCTCCAGGTGCATCCCC
  			GGGTACCAGCTCTACCGGTTCT
  			CCAGGTACTCCTGGCAGCGGTA
  			CTGCATCTTCCTCTCCAGGTGCT
  			TCTCCGGGCACCAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCGGG
  			CACTAGCTCTACTGGTTCTCCA
  			GGTGCATCCCCTGGCACTAGCT
  			CTACTGGTTCTCCAGGTGCTTCT
  			CCTGGTACCAGCTCTACTGGTT
  			CTCCAGGTACCCCTGGTAGCGG
  			TACTGCTTCTTCCTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCTACC
  			GGTTCTCCAGGTACCCCGGGTA
  			GCGGTACCGCATCTTCTTCTCCA
  			GGTAGCTCTACCCCGTCTGGTG
  			CTACTGGTTCTCCAGGTACTCC
  			GGGCAGCGGTACTGCTTCTTCC
  			TCTCCAGGTAGCTCTACCCCTTC
  			TGGTGCTACTGGCTCTCCAGGT
  			AGCTCTACCCCGTCTGGTGCTA
  			CTGGCTCCCCAGGTTCTAGCCC
  			TTCTGCATCCACCGGTACCGGT
  			CCAGGTTCTAGCCCGTCTGCAT
  			CTACTGGTACTGGTCCAGGTGC
  			ATCCCCGGGCACTAGCTCTACC
  			GGTTCTCCAGGTACTCCTGGTA
  			GCGGTACTGCTTCTTCTTCTCCA
  			GGTAGCTCTACTCCTTCTGGTGC
  			TACTGGTTCTCCAGGTTCTAGCC
  			CTTCTGCATCCACCGGTACCGG
  			CCCAGGTTCTAGCCCGTCTGCTT
  			CTACCGGTACTGGTCCAGGTGC
  			TTCTCCGGGTACTAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCTGGT
  			ACTAGCTCTACTGGTTCTCCAG
  			GTAGCTCTACTCCGTCTGGTGC
  			AACCGGCTCTCCAGGTTCTAGC
  			CCTTCTGCATCTACCGGTACTG
  			GTCCAGGTGCATCCCCTGGTAC
  			CAGCTCTACCGGTTCTCCAGGT
  			TCTAGCCCTTCTGCTTCTACCGG
  			TACCGGTCCAGGTACCCCTGGC
  			AGCGGTACCGCATCTTCCTCTC
  			CAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGTTCCCCAGGTAGC
  			TCTACTCCTTCTGGTGCTACTGG
  			CTCCCCAGGTGCATCCCCTGGC
  			ACCAGCTCTACCGGTTCTCCA
  FVII-	ANAFLEELRPGSLE	682	GCCAACGCGTTCCTGGAGGAGC	683
  AM875	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GTSTEPSEGSAPGS		AGGTCATCATCCCCAGCACGTA
  	EPATSGSETPGSPA		CGTCCCGGGCACCACCAACCAC
  	GSPTSTEEGSTSST		GACATCGCGCTGCTCCGCCTGC
  	AESPGPGTSTPESG		ACCAGCCCGTGGTCCTCACTGA
  	SASPGSTSESPSGT		CCATGTGGTGCCCCTCTGCCTG
  	APGSTSESPSGTAP		CCCGAACGGACGTTCTCTGAGA
  	GTSTPESGSASPGT		GGACGCTGGCCTTCGTGCGCTT
  	STPESGSASPGSEP		CTCATTGGTCAGCGGCTGGGGC
  	ATSGSETPGTSESA		CAGCTGCTGGACCGTGGCGCCA
  	TPESGPGSPAGSPT		CGGCCCTGGAGCTCATGGTCCT
  	STEEGTSTEPSEGS		CAACGTGCCCCGGCTGATGACC
  	APGTSESATPESGP		CAGGACTGCCTGCAGCAGTCAC
  	GTSTEPSEGSAPGT		GGAAGGTGGGAGACTCCCCAA
  	STEPSEGSAPGSPA		ATATCACGGAGTACATGTTCTG
  	GSPTSTEEGTSTEP		TGCCGGCTACTCGGATGGCAGC
  	SEGSAPGTSTEPSE		AAGGACTCCTGCAAGGGGGAC
  	GSAPGTSESATPES		AGTGGAGGCCCACATGCCACCC
  	GPGTSESATPESGP		ACTACCGGGGCACGTGGTACCT
  	GTSTEPSEGSAPGT		GACGGGCATCGTCAGCTGGGGC
  	STEPSEGSAPGTSE		CAGGGCTGCGCAACCGTGGGCC
  	SATPESGPGTSTEP		ACTTTGGGGTGTACACCAGGGT
  	SEGSAPGSEPATSG		CTCCCAGTACATCGAGTGGCTG
  	SETPGSPAGSPTST		CAAAAGCTCATGCGCTCAGAGC
  	EEGSSTPSGATGSP		CACGCCCAGGAGTCCTCCTGCG
  	GTPGSGTASSSPGS		AGCCCCATTTCCCGGTGGTACT
  	STPSGATGSPGTST		TCTACTGAACCGTCTGAAGGCA
  	EPSEGSAPGTSTEP		GCGCACCAGGTAGCGAACCGGC
  	SEGSAPGSEPATSG		TACTTCCGGTTCTGAAACCCCA
  	SETPGSPAGSPTST		GGTAGCCCAGCAGGTTCTCCAA
  	EEGSPAGSPTSTEE		CTTCTACTGAAGAAGGTTCTAC
  	GTSTEPSEGSAPGA		CAGCTCTACCGCAGAATCTCCT
  	SASGAPSTGGTSES		GGTCCAGGTACCTCTACTCCGG
  	ATPESGPGSPAGSP		AAAGCGGCTCTGCATCTCCAGG
  	TSTEEGSPAGSPTS		TTCTACTAGCGAATCTCCTTCTG
  	TEEGSTSSTAESPG		GCACTGCACCAGGTTCTACTAG
  	PGSTSESPSGTAPG		CGAATCCCCGTCTGGTACTGCT
  	TSPSGESSTAPGTP		CCAGGTACTTCTACTCCTGAAA
  	GSGTASSSPGSSTP		GCGGTTCCGCTTCTCCAGGTAC
  	SGATGSPGSSPSAS		CTCTACTCCGGAAAGCGGTTCT
  	TGTGPGSEPATSGS		GCATCTCCAGGTAGCGAACCGG
  	ETPGTSESATPESG		CAACCTCCGGCTCTGAAACCCC
  	PGSEPATSGSETPG		AGGTACCTCTGAAAGCGCTACT
  	STSSTAESPGPGST		CCTGAATCCGGCCCAGGTAGCC
  	SSTAESPGPGTSPS		CGGCAGGTTCTCCGACTTCCAC
  	GESSTAPGSEPATS		TGAGGAAGGTACCTCTACTGAA
  	GSETPGSEPATSGS		CCTTCTGAGGGCAGCGCTCCAG
  	ETPGTSTEPSEGSA		GTACTTCTGAAAGCGCTACCCC
  	PGSTSSTAESPGPG		GGAGTCCGGTCCAGGTACTTCT
  	TSTPESGSASPGST		ACTGAACCGTCCGAAGGTAGCG
  	SESPSGTAPGTSTE		CACCAGGTACTTCTACCGAACC
  	PSEGSAPGTSTEPS		GTCCGAGGGTAGCGCACCAGGT
  	EGSAPGTSTEPSEG		AGCCCAGCAGGTTCTCCTACCT
  	SAPGSSTPSGATGS		CCACCGAGGAAGGTACTTCTAC
  	PGSSPSASTGTGPG		CGAACCGTCCGAGGGTAGCGCA
  	ASPGTSSTGSPGSE		CCAGGTACTTCTACCGAACCTT
  	PATSGSETPGTSES		CCGAGGGCAGCGCACCAGGTAC
  	ATPESGPGSPAGSP		TTCTGAAAGCGCTACCCCTGAG
  	TSTEEGSSTPSGAT		TCCGGCCCAGGTACTTCTGAAA
  	GSPGSSPSASTGTG		GCGCTACTCCTGAATCCGGTCC
  	PGASPGTSSTGSPG		AGGTACCTCTACTGAACCTTCC
  	TSESATPESGPGTS		GAAGGCAGCGCTCCAGGTACCT
  	TEPSEGSAPGTSTE		CTACCGAACCGTCCGAGGGCAG
  	PSEGSAP		CGCACCAGGTACTTCTGAAAGC
  			GCAACCCCTGAATCCGGTCCAG
  			GTACTTCTACTGAACCTTCCGA
  			AGGTAGCGCTCCAGGTAGCGAA
  			CCTGCTACTTCTGGTTCTGAAAC
  			CCCAGGTAGCCCGGCTGGCTCT
  			CCGACCTCCACCGAGGAAGGTA
  			GCTCTACCCCGTCTGGTGCTACT
  			GGTTCTCCAGGTACTCCGGGCA
  			GCGGTACTGCTTCTTCCTCTCCA
  			GGTAGCTCTACCCCTTCTGGTG
  			CTACTGGCTCTCCAGGTACCTCT
  			ACCGAACCGTCCGAGGGTAGCG
  			CACCAGGTACCTCTACTGAACC
  			GTCTGAGGGTAGCGCTCCAGGT
  			AGCGAACCGGCAACCTCCGGTT
  			CTGAAACTCCAGGTAGCCCTGC
  			TGGCTCTCCGACTTCTACTGAG
  			GAAGGTAGCCCGGCTGGTTCTC
  			CGACTTCTACTGAGGAAGGTAC
  			TTCTACCGAACCTTCCGAAGGT
  			AGCGCTCCAGGTGCAAGCGCAA
  			GCGGCGCGCCAAGCACGGGAG
  			GTACTTCTGAAAGCGCTACTCC
  			TGAGTCCGGCCCAGGTAGCCCG
  			GCTGGCTCTCCGACTTCCACCG
  			AGGAAGGTAGCCCGGCTGGCTC
  			TCCAACTTCTACTGAAGAAGGT
  			TCTACCAGCTCTACCGCTGAAT
  			CTCCTGGCCCAGGTTCTACTAG
  			CGAATCTCCGTCTGGCACCGCA
  			CCAGGTACTTCCCCTAGCGGTG
  			AATCTTCTACTGCACCAGGTAC
  			CCCTGGCAGCGGTACCGCTTCT
  			TCCTCTCCAGGTAGCTCTACCCC
  			GTCTGGTGCTACTGGCTCTCCA
  			GGTTCTAGCCCGTCTGCATCTA
  			CCGGTACCGGCCCAGGTAGCGA
  			ACCGGCAACCTCCGGCTCTGAA
  			ACTCCAGGTACTTCTGAAAGCG
  			CTACTCCGGAATCCGGCCCAGG
  			TAGCGAACCGGCTACTTCCGGC
  			TCTGAAACCCCAGGTTCCACCA
  			GCTCTACTGCAGAATCTCCGGG
  			CCCAGGTTCTACTAGCTCTACT
  			GCAGAATCTCCGGGTCCAGGTA
  			CTTCTCCTAGCGGCGAATCTTCT
  			ACCGCTCCAGGTAGCGAACCGG
  			CAACCTCTGGCTCTGAAACTCC
  			AGGTAGCGAACCTGCAACCTCC
  			GGCTCTGAAACCCCAGGTACTT
  			CTACTGAACCTTCTGAGGGCAG
  			CGCACCAGGTTCTACCAGCTCT
  			ACCGCAGAATCTCCTGGTCCAG
  			GTACCTCTACTCCGGAAAGCGG
  			CTCTGCATCTCCAGGTTCTACTA
  			GCGAATCTCCTTCTGGCACTGC
  			ACCAGGTACTTCTACCGAACCG
  			TCCGAAGGCAGCGCTCCAGGTA
  			CCTCTACTGAACCTTCCGAGGG
  			CAGCGCTCCAGGTACCTCTACC
  			GAACCTTCTGAAGGTAGCGCAC
  			CAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGCTCCCCAGGTTCT
  			AGCCCGTCTGCTTCCACTGGTA
  			CTGGCCCAGGTGCTTCCCCGGG
  			CACCAGCTCTACTGGTTCTCCA
  			GGTAGCGAACCTGCTACCTCCG
  			GTTCTGAAACCCCAGGTACCTC
  			TGAAAGCGCAACTCCGGAGTCT
  			GGTCCAGGTAGCCCTGCAGGTT
  			CTCCTACCTCCACTGAGGAAGG
  			TAGCTCTACTCCGTCTGGTGCA
  			ACCGGCTCCCCAGGTTCTAGCC
  			CGTCTGCTTCCACTGGTACTGG
  			CCCAGGTGCTTCCCCGGGCACC
  			AGCTCTACTGGTTCTCCAGGTA
  			CCTCTGAAAGCGCTACTCCGGA
  			GTCTGGCCCAGGTACCTCTACT
  			GAACCGTCTGAGGGTAGCGCTC
  			CAGGTACTTCTACTGAACCGTC
  			CGAAGGTAGCGCACCA
  FVII-	ANAFLEELRPGSLE	684	GCCAACGCGTTCCTGGAGGAGC	685
  AM1318	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	GTSTEPSEGSAPGS		AGGTCATCATCCCCAGCACGTA
  	EPATSGSETPGSPA		CGTCCCGGGCACCACCAACCAC
  	GSPTSTEEGSTSST		GACATCGCGCTGCTCCGCCTGC
  	AESPGPGTSTPESG		ACCAGCCCGTGGTCCTCACTGA
  	SASPGSTSESPSGT		CCATGTGGTGCCCCTCTGCCTG
  	APGSTSESPSGTAP		CCCGAACGGACGTTCTCTGAGA
  	GTSTPESGSASPGT		GGACGCTGGCCTTCGTGCGCTT
  	STPESGSASPGSEP		CTCATTGGTCAGCGGCTGGGGC
  	ATSGSETPGTSESA		CAGCTGCTGGACCGTGGCGCCA
  	TPESGPGSPAGSPT		CGGCCCTGGAGCTCATGGTCCT
  	STEEGTSTEPSEGS		CAACGTGCCCCGGCTGATGACC
  	APGTSESATPESGP		CAGGACTGCCTGCAGCAGTCAC
  	GTSTEPSEGSAPGT		GGAAGGTGGGAGACTCCCCAA
  	STEPSEGSAPGSPA		ATATCACGGAGTACATGTTCTG
  	GSPTSTEEGTSTEP		TGCCGGCTACTCGGATGGCAGC
  	SEGSAPGTSTEPSE		AAGGACTCCTGCAAGGGGGAC
  	GSAPGTSESATPES		AGTGGAGGCCCACATGCCACCC
  	GPGTSESATPESGP		ACTACCGGGGCACGTGGTACCT
  	GTSTEPSEGSAPGT		GACGGGCATCGTCAGCTGGGGC
  	STEPSEGSAPGTSE		CAGGGCTGCGCAACCGTGGGCC
  	SATPESGPGTSTEP		ACTTTGGGGTGTACACCAGGGT
  	SEGSAPGSEPATSG		CTCCCAGTACATCGAGTGGCTG
  	SETPGSPAGSPTST		CAAAAGCTCATGCGCTCAGAGC
  	EEGSSTPSGATGSP		CACGCCCAGGAGTCCTCCTGCG
  	GTPGSGTASSSPGS		AGCCCCATTTCCCGGTGGTACT
  	STPSGATGSPGTST		TCTACTGAACCGTCTGAAGGCA
  	EPSEGSAPGTSTEP		GCGCACCAGGTAGCGAACCGGC
  	SEGSAPGSEPATSG		TACTTCCGGTTCTGAAACCCCA
  	SETPGSPAGSPTST		GGTAGCCCAGCAGGTTCTCCAA
  	EEGSPAGSPTSTEE		CTTCTACTGAAGAAGGTTCTAC
  	GTSTEPSEGSAPGP		CAGCTCTACCGCAGAATCTCCT
  	EPTGPAPSGGSEPA		GGTCCAGGTACCTCTACTCCGG
  	TSGSETPGTSESAT		AAAGCGGCTCTGCATCTCCAGG
  	PESGPGSPAGSPTS		TTCTACTAGCGAATCTCCTTCTG
  	TEEGTSESATPESG		GCACTGCACCAGGTTCTACTAG
  	PGSPAGSPTSTEEG		CGAATCCCCGTCTGGTACTGCT
  	SPAGSPTSTEEGTS		CCAGGTACTTCTACTCCTGAAA
  	ESATPESGPGSPAG		GCGGTTCCGCTTCTCCAGGTAC
  	SPTSTEEGSPAGSP		CTCTACTCCGGAAAGCGGTTCT
  	TSTEEGSTSSTAES		GCATCTCCAGGTAGCGAACCGG
  	PGPGSTSESPSGTA		CAACCTCCGGCTCTGAAACCCC
  	PGTSPSGESSTAPG		AGGTACCTCTGAAAGCGCTACT
  	STSESPSGTAPGST		CCTGAATCCGGCCCAGGTAGCC
  	SESPSGTAPGTSPS		CGGCAGGTTCTCCGACTTCCAC
  	GESSTAPGTSTEPS		TGAGGAAGGTACCTCTACTGAA
  	EGSAPGTSESATPE		CCTTCTGAGGGCAGCGCTCCAG
  	SGPGTSESATPESG		GTACTTCTGAAAGCGCTACCCC
  	PGSEPATSGSETPG		GGAGTCCGGTCCAGGTACTTCT
  	TSESATPESGPGTS		ACTGAACCGTCCGAAGGTAGCG
  	ESATPESGPGTSTE		CACCAGGTACTTCTACCGAACC
  	PSEGSAPGTSESAT		GTCCGAGGGTAGCGCACCAGGT
  	PESGPGTSTEPSEG		AGCCCAGCAGGTTCTCCTACCT
  	SAPGTSPSGESSTA		CCACCGAGGAAGGTACTTCTAC
  	PGTSPSGESSTAPG		CGAACCGTCCGAGGGTAGCGCA
  	TSPSGESSTAPGTS		CCAGGTACTTCTACCGAACCTT
  	TEPSEGSAPGSPAG		CCGAGGGCAGCGCACCAGGTAC
  	SPTSTEEGTSTEPS		TTCTGAAAGCGCTACCCCTGAG
  	EGSAPGSSPSASTG		TCCGGCCCAGGTACTTCTGAAA
  	TGPGSSTPSGATGS		GCGCTACTCCTGAATCCGGTCC
  	PGSSTPSGATGSPG		AGGTACCTCTACTGAACCTTCC
  	SSTPSGATGSPGSS		GAAGGCAGCGCTCCAGGTACCT
  	TPSGATGSPGASPG		CTACCGAACCGTCCGAGGGCAG
  	TSSTGSPGASASGA		CGCACCAGGTACTTCTGAAAGC
  	PSTGGTSPSGESST		GCAACCCCTGAATCCGGTCCAG
  	APGSTSSTAESPGP		GTACTTCTACTGAACCTTCCGA
  	GTSPSGESSTAPGT		AGGTAGCGCTCCAGGTAGCGAA
  	SESATPESGPGTST		CCTGCTACTTCTGGTTCTGAAAC
  	EPSEGSAPGTSTEP		CCCAGGTAGCCCGGCTGGCTCT
  	SEGSAPGSSPSAST		CCGACCTCCACCGAGGAAGGTA
  	GTGPGSSTPSGAT		GCTCTACCCCGTCTGGTGCTACT
  	GSPGASPGTSSTGS		GGTTCTCCAGGTACTCCGGGCA
  	PGTSTPESGSASPG		GCGGTACTGCTTCTTCCTCTCCA
  	TSPSGESSTAPGTS		GGTAGCTCTACCCCTTCTGGTG
  	PSGESSTAPGTSES		CTACTGGCTCTCCAGGTACCTCT
  	ATPESGPGSEPATS		ACCGAACCGTCCGAGGGTAGCG
  	GSETPGTSTEPSEG		CACCAGGTACCTCTACTGAACC
  	SAPGSTSESPSGTA		GTCTGAGGGTAGCGCTCCAGGT
  	PGSTSESPSGTAPG		AGCGAACCGGCAACCTCCGGTT
  	TSTPESGSASPGSP		CTGAAACTCCAGGTAGCCCTGC
  	AGSPTSTEEGTSES		TGGCTCTCCGACTTCTACTGAG
  	ATPESGPGTSTEPS		GAAGGTAGCCCGGCTGGTTCTC
  	EGSAPGSPAGSPTS		CGACTTCTACTGAGGAAGGTAC
  	TEEGTSESATPESG		TTCTACCGAACCTTCCGAAGGT
  	PGSEPATSGSETPG		AGCGCTCCAGGTCCAGAACCAA
  	SSTPSGATGSPGAS		CGGGGCCGGCCCCAAGCGGAG
  	PGTSSTGSPGSSTP		GTAGCGAACCGGCAACCTCCGG
  	SGATGSPGSTSESP		CTCTGAAACCCCAGGTACCTCT
  	SGTAPGTSPSGESS		GAAAGCGCTACTCCTGAATCCG
  	TAPGSTSSTAESPG		GCCCAGGTAGCCCGGCAGGTTC
  	PGSSTPSGATGSPG		TCCGACTTCCACTGAGGAAGGT
  	ASPGTSSTGSPGTP		ACTTCTGAAAGCGCTACTCCTG
  	GSGTASSSPGSPAG		AGTCCGGCCCAGGTAGCCCGGC
  	SPTSTEEGSPAGSP		TGGCTCTCCGACTTCCACCGAG
  	TSTEEGTSTEPSEG		GAAGGTAGCCCGGCTGGCTCTC
  	SAP		CAACTTCTACTGAAGAAGGTAC
  			TTCTGAAAGCGCTACTCCTGAG
  			TCCGGCCCAGGTAGCCCGGCTG
  			GCTCTCCGACTTCCACCGAGGA
  			AGGTAGCCCGGCTGGCTCTCCA
  			ACTTCTACTGAAGAAGGTTCTA
  			CCAGCTCTACCGCTGAATCTCC
  			TGGCCCAGGTTCTACTAGCGAA
  			TCTCCGTCTGGCACCGCACCAG
  			GTACTTCCCCTAGCGGTGAATC
  			TTCTACTGCACCAGGTTCTACC
  			AGCGAATCTCCTTCTGGCACCG
  			CTCCAGGTTCTACTAGCGAATC
  			CCCGTCTGGTACCGCACCAGGT
  			ACTTCTCCTAGCGGCGAATCTT
  			CTACCGCACCAGGTACTTCTAC
  			CGAACCTTCCGAGGGCAGCGCA
  			CCAGGTACTTCTGAAAGCGCTA
  			CCCCTGAGTCCGGCCCAGGTAC
  			TTCTGAAAGCGCTACTCCTGAA
  			TCCGGTCCAGGTAGCGAACCGG
  			CAACCTCTGGCTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAATCTGGTCCAGGTACTT
  			CTGAAAGCGCTACTCCGGAATC
  			CGGTCCAGGTACCTCTACTGAA
  			CCTTCTGAGGGCAGCGCTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAGTCCGGTCCAGGTACTTCT
  			ACTGAACCGTCCGAAGGTAGCG
  			CACCAGGTACCTCCCCTAGCGG
  			CGAATCTTCTACTGCTCCAGGT
  			ACCTCTCCTAGCGGCGAATCTT
  			CTACCGCTCCAGGTACCTCCCC
  			TAGCGGTGAATCTTCTACCGCA
  			CCAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAG
  			CCCAGCAGGTTCTCCTACCTCC
  			ACCGAGGAAGGTACTTCTACCG
  			AACCGTCCGAGGGTAGCGCACC
  			AGGTTCTAGCCCTTCTGCTTCCA
  			CCGGTACCGGCCCAGGTAGCTC
  			TACTCCGTCTGGTGCAACTGGC
  			TCTCCAGGTAGCTCTACTCCGTC
  			TGGTGCAACCGGCTCCCCAGGT
  			AGCTCTACCCCGTCTGGTGCTA
  			CCGGCTCTCCAGGTAGCTCTAC
  			CCCGTCTGGTGCAACCGGCTCC
  			CCAGGTGCATCCCCGGGTACTA
  			GCTCTACCGGTTCTCCAGGTGC
  			AAGCGCAAGCGGCGCGCCAAG
  			CACGGGAGGTACTTCTCCGAGC
  			GGTGAATCTTCTACCGCACCAG
  			GTTCTACTAGCTCTACCGCTGA
  			ATCTCCGGGCCCAGGTACTTCT
  			CCGAGCGGTGAATCTTCTACTG
  			CTCCAGGTACCTCTGAAAGCGC
  			TACTCCGGAGTCTGGCCCAGGT
  			ACCTCTACTGAACCGTCTGAGG
  			GTAGCGCTCCAGGTACTTCTAC
  			TGAACCGTCCGAAGGTAGCGCA
  			CCAGGTTCTAGCCCTTCTGCATC
  			TACTGGTACTGGCCCAGGTAGC
  			TCTACTCCTTCTGGTGCTACCGG
  			CTCTCCAGGTGCTTCTCCGGGT
  			ACTAGCTCTACCGGTTCTCCAG
  			GTACTTCTACTCCGGAAAGCGG
  			TTCCGCATCTCCAGGTACTTCTC
  			CTAGCGGTGAATCTTCTACTGC
  			TCCAGGTACCTCTCCTAGCGGC
  			GAATCTTCTACTGCTCCAGGTA
  			CTTCTGAAAGCGCAACCCCTGA
  			ATCCGGTCCAGGTAGCGAACCG
  			GCTACTTCTGGCTCTGAGACTC
  			CAGGTACTTCTACCGAACCGTC
  			CGAAGGTAGCGCACCAGGTTCT
  			ACCAGCGAATCCCCTTCTGGTA
  			CTGCTCCAGGTTCTACCAGCGA
  			ATCCCCTTCTGGCACCGCACCA
  			GGTACTTCTACCCCTGAAAGCG
  			GCTCCGCTTCTCCAGGTAGCCC
  			GGCAGGCTCTCCGACCTCTACT
  			GAGGAAGGTACTTCTGAAAGCG
  			CAACCCCGGAGTCCGGCCCAGG
  			TACCTCTACCGAACCGTCTGAG
  			GGCAGCGCACCAGGTAGCCCTG
  			CTGGCTCTCCAACCTCCACCGA
  			AGAAGGTACCTCTGAAAGCGCA
  			ACCCCTGAATCCGGCCCAGGTA
  			GCGAACCGGCAACCTCCGGTTC
  			TGAAACCCCAGGTAGCTCTACC
  			CCGTCTGGTGCTACCGGTTCCC
  			CAGGTGCTTCTCCTGGTACTAG
  			CTCTACCGGTTCTCCAGGTAGC
  			TCTACCCCGTCTGGTGCTACTG
  			GCTCTCCAGGTTCTACTAGCGA
  			ATCCCCGTCTGGTACTGCTCCA
  			GGTACTTCCCCTAGCGGTGAAT
  			CTTCTACTGCTCCAGGTTCTACC
  			AGCTCTACCGCAGAATCTCCGG
  			GTCCAGGTAGCTCTACCCCTTCT
  			GGTGCAACCGGCTCTCCAGGTG
  			CATCCCCGGGTACCAGCTCTAC
  			CGGTTCTCCAGGTACTCCGGGT
  			AGCGGTACCGCTTCTTCCTCTCC
  			AGGTAGCCCTGCTGGCTCTCCG
  			ACTTCTACTGAGGAAGGTAGCC
  			CGGCTGGTTCTCCGACTTCTACT
  			GAGGAAGGTACTTCTACCGAAC
  			CTTCCGAAGGTAGCGCTCCA
  FIX-	YNSGKLEEFVQGN	686	TATAATTCAGGTAAATTGGAAG	687
  AE288	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	TSESATPESGPGSE		CATAATATTGAGGAGACAGAAC
  	PATSGSETPGTSES		ATACAGAGCAAAAGCGAAATG
  	ATPESGPGSEPATS		TGATTCGAATTATTCCTCACCAC
  	GSETPGTSESATPE		AACTACAATGCAGCTATTAATA
  	SGPGTSTEPSEGSA		AGTACAACCATGACATTGCCCT
  	PGSPAGSPTSTEEG		TCTGGAACTGGACGAACCCTTA
  	TSESATPESGPGSE		GTGCTAAACAGCTACGTTACAC
  	PATSGSETPGTSES		CTATTTGCATTGCTGACAAGGA
  	ATPESGPGSPAGSP		ATACACGAACATCTTCCTCAAA
  	TSTEEGSPAGSPTS		TTTGGATCTGGCTATGTAAGTG
  	TEEGTSTEPSEGSA		GCTGGGGAAGAGTCTTCCACAA
  	PGTSESATPESGPG		AGGGAGATCAGCTTTAGTTCTT
  	TSESATPESGPGTS		CAGTACCTTAGAGTTCCACTTG
  	ESATPESGPGSEPA		TTGACCGAGCCACATGTCTTCG
  	TSGSETPGSEPATS		ATCTACAAAGTTCACCATCTAT
  	GSETPGSPAGSPTS		AACAACATGTTCTGTGCTGGCT
  	TEEGTSTEPSEGSA		TCCATGAAGGAGGTAGAGATTC
  	PGTSTEPSEGSAPG		ATGTCAAGGAGATAGTGGGGG
  	SEPATSGSETPGTS		ACCCCATGTTACTGAAGTGGAA
  	ESATPESGPGTSTE		GGGACCAGTTTCTTAACTGGAA
  	PSEGSAP		TTATTAGCTGGGGTGAAGAGTG
  			TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGTGGTA
  			CCTCTGAAAGCGCAACTCCTGA
  			GTCTGGCCCAGGTAGCGAACCT
  			GCTACCTCCGGCTCTGAGACTC
  			CAGGTACCTCTGAAAGCGCAAC
  			CCCGGAATCTGGTCCAGGTAGC
  			GAACCTGCAACCTCTGGCTCTG
  			AAACCCCAGGTACCTCTGAAAG
  			CGCTACTCCTGAATCTGGCCCA
  			GGTACTTCTACTGAACCGTCCG
  			AGGGCAGCGCACCAGGTAGCCC
  			TGCTGGCTCTCCAACCTCCACC
  			GAAGAAGGTACCTCTGAAAGCG
  			CAACCCCTGAATCCGGCCCAGG
  			TAGCGAACCGGCAACCTCCGGT
  			TCTGAAACCCCAGGTACTTCTG
  			AAAGCGCTACTCCTGAGTCCGG
  			CCCAGGTAGCCCGGCTGGCTCT
  			CCGACTTCCACCGAGGAAGGTA
  			GCCCGGCTGGCTCTCCAACTTC
  			TACTGAAGAAGGTACTTCTACC
  			GAACCTTCCGAGGGCAGCGCAC
  			CAGGTACTTCTGAAAGCGCTAC
  			CCCTGAGTCCGGCCCAGGTACT
  			TCTGAAAGCGCTACTCCTGAAT
  			CCGGTCCAGGTACTTCTGAAAG
  			CGCTACCCCGGAATCTGGCCCA
  			GGTAGCGAACCGGCTACTTCTG
  			GTTCTGAAACCCCAGGTAGCGA
  			ACCGGCTACCTCCGGTTCTGAA
  			ACTCCAGGTAGCCCAGCAGGCT
  			CTCCGACTTCCACTGAGGAAGG
  			TACTTCTACTGAACCTTCCGAA
  			GGCAGCGCACCAGGTACCTCTA
  			CTGAACCTTCTGAGGGCAGCGC
  			TCCAGGTAGCGAACCTGCAACC
  			TCTGGCTCTGAAACCCCAGGTA
  			CCTCTGAAAGCGCTACTCCTGA
  			ATCTGGCCCAGGTACTTCTACT
  			GAACCGTCCGAGGGCAGCGCAC
  			CA
  FIX-	YNSGKLEEFVQGN	688	TATAATTCAGGTAAATTGGAAG	689
  AE864	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	SPAGSPTSTEEGTS		CATAATATTGAGGAGACAGAAC
  	ESATPESGPGTSTE		ATACAGAGCAAAAGCGAAATG
  	PSEGSAPGSPAGSP		TGATTCGAATTATTCCTCACCAC
  	TSTEEGTSTEPSEG		AACTACAATGCAGCTATTAATA
  	SAPGTSTEPSEGSA		AGTACAACCATGACATTGCCCT
  	PGTSESATPESGPG		TCTGGAACTGGACGAACCCTTA
  	SEPATSGSETPGSE		GTGCTAAACAGCTACGTTACAC
  	PATSGSETPGSPAG		CTATTTGCATTGCTGACAAGGA
  	SPTSTEEGTSESAT		ATACACGAACATCTTCCTCAAA
  	PESGPGTSTEPSEG		TTTGGATCTGGCTATGTAAGTG
  	SAPGTSTEPSEGSA		GCTGGGGAAGAGTCTTCCACAA
  	PGSPAGSPTSTEEG		AGGGAGATCAGCTTTAGTTCTT
  	TSTEPSEGSAPGTS		CAGTACCTTAGAGTTCCACTTG
  	TEPSEGSAPGTSES		TTGACCGAGCCACATGTCTTCG
  	ATPESGPGTSTEPS		ATCTACAAAGTTCACCATCTAT
  	EGSAPGTSESATPE		AACAACATGTTCTGTGCTGGCT
  	SGPGSEPATSGSET		TCCATGAAGGAGGTAGAGATTC
  	PGTSTEPSEGSAPG		ATGTCAAGGAGATAGTGGGGG
  	TSTEPSEGSAPGTS		ACCCCATGTTACTGAAGTGGAA
  	ESATPESGPGTSES		GGGACCAGTTTCTTAACTGGAA
  	ATPESGPGSPAGSP		TTATTAGCTGGGGTGAAGAGTG
  	TSTEEGTSESATPE		TGCAATGAAAGGCAAATATGGA
  	SGPGSEPATSGSET		ATATATACCAAGGTATCCCGGT
  	PGTSESATPESGPG		ATGTCAACTGGATTAAGGAAAA
  	TSTEPSEGSAPGTS		AACAAAGCTCACTGGGGTGGTA
  	TEPSEGSAPGTSTE		GCCCGGCTGGCTCTCCTACCTCT
  	PSEGSAPGTSTEPS		ACTGAGGAAGGTACTTCTGAAA
  	EGSAPGTSTEPSEG		GCGCTACTCCTGAGTCTGGTCC
  	SAPGTSTEPSEGSA		AGGTACCTCTACTGAACCGTCC
  	PGSPAGSPTSTEEG		GAAGGTAGCGCTCCAGGTAGCC
  	TSTEPSEGSAPGTS		CAGCAGGCTCTCCGACTTCCAC
  	ESATPESGPGSEPA		TGAGGAAGGTACTTCTACTGAA
  	TSGSETPGTSESAT		CCTTCCGAAGGCAGCGCACCAG
  	PESGPGSEPATSGS		GTACCTCTACTGAACCTTCTGA
  	ETPGTSESATPESG		GGGCAGCGCTCCAGGTACTTCT
  	PGTSTEPSEGSAPG		GAAAGCGCTACCCCGGAATCTG
  	TSESATPESGPGSP		GCCCAGGTAGCGAACCGGCTAC
  	AGSPTSTEEGSPAG		TTCTGGTTCTGAAACCCCAGGT
  	SPTSTEEGSPAGSP		AGCGAACCGGCTACCTCCGGTT
  	TSTEEGTSESATPE		CTGAAACTCCAGGTAGCCCGGC
  	SGPGTSTEPSEGSA		AGGCTCTCCGACCTCTACTGAG
  	PGTSESATPESGPG		GAAGGTACTTCTGAAAGCGCAA
  	SEPATSGSETPGTS		CCCCGGAGTCCGGCCCAGGTAC
  	ESATPESGPGSEPA		CTCTACCGAACCGTCTGAGGGC
  	TSGSETPGTSESAT		AGCGCACCAGGTACTTCTACCG
  	PESGPGTSTEPSEG		AACCGTCCGAGGGTAGCGCACC
  	SAPGSPAGSPTSTE		AGGTAGCCCAGCAGGTTCTCCT
  	EGTSESATPESGPG		ACCTCCACCGAGGAAGGTACTT
  	SEPATSGSETPGTS		CTACCGAACCGTCCGAGGGTAG
  	ESATPESGPGSPAG		CGCACCAGGTACCTCTACTGAA
  	SPTSTEEGSPAGSP		CCTTCTGAGGGCAGCGCTCCAG
  	TSTEEGTSTEPSEG		GTACTTCTGAAAGCGCTACCCC
  	SAPGTSESATPESG		GGAGTCCGGTCCAGGTACTTCT
  	PGTSESATPESGPG		ACTGAACCGTCCGAAGGTAGCG
  	TSESATPESGPGSE		CACCAGGTACTTCTGAAAGCGC
  	PATSGSETPGSEPA		AACCCCTGAATCCGGTCCAGGT
  	TSGSETPGSPAGSP		AGCGAACCGGCTACTTCTGGCT
  	TSTEEGTSTEPSEG		CTGAGACTCCAGGTACTTCTAC
  	SAPGTSTEPSEGSA		CGAACCGTCCGAAGGTAGCGCA
  	PGSEPATSGSETPG		CCAGGTACTTCTACTGAACCGT
  	TSESATPESGPGTS		CTGAAGGTAGCGCACCAGGTAC
  	TEPSEGSAP		TTCTGAAAGCGCAACCCCGGAA
  			TCCGGCCCAGGTACCTCTGAAA
  			GCGCAACCCCGGAGTCCGGCCC
  			AGGTAGCCCTGCTGGCTCTCCA
  			ACCTCCACCGAAGAAGGTACCT
  			CTGAAAGCGCAACCCCTGAATC
  			CGGCCCAGGTAGCGAACCGGCA
  			ACCTCCGGTTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			GGAGTCTGGCCCAGGTACCTCT
  			ACTGAACCGTCTGAGGGTAGCG
  			CTCCAGGTACTTCTACTGAACC
  			GTCCGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAAG
  			GCAGCGCTCCAGGTACCTCTAC
  			TGAACCTTCCGAGGGCAGCGCT
  			CCAGGTACCTCTACCGAACCTT
  			CTGAAGGTAGCGCACCAGGTAC
  			TTCTACCGAACCGTCCGAGGGT
  			AGCGCACCAGGTAGCCCAGCAG
  			GTTCTCCTACCTCCACCGAGGA
  			AGGTACTTCTACCGAACCGTCC
  			GAGGGTAGCGCACCAGGTACCT
  			CTGAAAGCGCAACTCCTGAGTC
  			TGGCCCAGGTAGCGAACCTGCT
  			ACCTCCGGCTCTGAGACTCCAG
  			GTACCTCTGAAAGCGCAACCCC
  			GGAATCTGGTCCAGGTAGCGAA
  			CCTGCAACCTCTGGCTCTGAAA
  			CCCCAGGTACCTCTGAAAGCGC
  			TACTCCTGAATCTGGCCCAGGT
  			ACTTCTACTGAACCGTCCGAGG
  			GCAGCGCACCAGGTACTTCTGA
  			AAGCGCTACTCCTGAGTCCGGC
  			CCAGGTAGCCCGGCTGGCTCTC
  			CGACTTCCACCGAGGAAGGTAG
  			CCCGGCTGGCTCTCCAACTTCT
  			ACTGAAGAAGGTAGCCCGGCA
  			GGCTCTCCGACCTCTACTGAGG
  			AAGGTACTTCTGAAAGCGCAAC
  			CCCGGAGTCCGGCCCAGGTACC
  			TCTACCGAACCGTCTGAGGGCA
  			GCGCACCAGGTACCTCTGAAAG
  			CGCAACTCCTGAGTCTGGCCCA
  			GGTAGCGAACCTGCTACCTCCG
  			GCTCTGAGACTCCAGGTACCTC
  			TGAAAGCGCAACCCCGGAATCT
  			GGTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCAGGTAGCCCTGCTGGCTCT
  			CCAACCTCCACCGAAGAAGGTA
  			CCTCTGAAAGCGCAACCCCTGA
  			ATCCGGCCCAGGTAGCGAACCG
  			GCAACCTCCGGTTCTGAAACCC
  			CAGGTACTTCTGAAAGCGCTAC
  			TCCTGAGTCCGGCCCAGGTAGC
  			CCGGCTGGCTCTCCGACTTCCA
  			CCGAGGAAGGTAGCCCGGCTGG
  			CTCTCCAACTTCTACTGAAGAA
  			GGTACTTCTACCGAACCTTCCG
  			AGGGCAGCGCACCAGGTACTTC
  			TGAAAGCGCTACCCCTGAGTCC
  			GGCCCAGGTACTTCTGAAAGCG
  			CTACTCCTGAATCCGGTCCAGG
  			TACTTCTGAAAGCGCTACCCCG
  			GAATCTGGCCCAGGTAGCGAAC
  			CGGCTACTTCTGGTTCTGAAAC
  			CCCAGGTAGCGAACCGGCTACC
  			TCCGGTTCTGAAACTCCAGGTA
  			GCCCAGCAGGCTCTCCGACTTC
  			CACTGAGGAAGGTACTTCTACT
  			GAACCTTCCGAAGGCAGCGCAC
  			CAGGTACCTCTACTGAACCTTC
  			TGAGGGCAGCGCTCCAGGTAGC
  			GAACCTGCAACCTCTGGCTCTG
  			AAACCCCAGGTACCTCTGAAAG
  			CGCTACTCCTGAATCTGGCCCA
  			GGTACTTCTACTGAACCGTCCG
  			AGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	690	TATAATTCAGGTAAATTGGAAG	691
  AF864	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	STSESPSGTAPGTS		CATAATATTGAGGAGACAGAAC
  	PSGESSTAPGSTSE		ATACAGAGCAAAAGCGAAATG
  	SPSGTAPGSTSESP		TGATTCGAATTATTCCTCACCAC
  	SGTAPGTSTPESGS		AACTACAATGCAGCTATTAATA
  	ASPGTSTPESGSAS		AGTACAACCATGACATTGCCCT
  	PGSTSESPSGTAPG		TCTGGAACTGGACGAACCCTTA
  	STSESPSGTAPGTS		GTGCTAAACAGCTACGTTACAC
  	PSGESSTAPGSTSE		CTATTTGCATTGCTGACAAGGA
  	SPSGTAPGTSPSGE		ATACACGAACATCTTCCTCAAA
  	SSTAPGTSPSGESS		TTTGGATCTGGCTATGTAAGTG
  	TAPGSTSSTAESPG		GCTGGGGAAGAGTCTTCCACAA
  	PGTSPSGESSTAPG		AGGGAGATCAGCTTTAGTTCTT
  	TSPSGESSTAPGST		CAGTACCTTAGAGTTCCACTTG
  	SSTAESPGPGTSTP		TTGACCGAGCCACATGTCTTCG
  	ESGSASPGTSTPES		ATCTACAAAGTTCACCATCTAT
  	GSASPGSTSESPSG		AACAACATGTTCTGTGCTGGCT
  	TAPGSTSESPSGTA		TCCATGAAGGAGGTAGAGATTC
  	PGTSTPESGSASPG		ATGTCAAGGAGATAGTGGGGG
  	STSSTAESPGPGTS		ACCCCATGTTACTGAAGTGGAA
  	TPESGSASPGSTSE		GGGACCAGTTTCTTAACTGGAA
  	SPSGTAPGTSPSGE		TTATTAGCTGGGGTGAAGAGTG
  	SSTAPGSTSSTAES		TGCAATGAAAGGCAAATATGGA
  	PGPGTSPSGESSTA		ATATATACCAAGGTATCCCGGT
  	PGTSTPESGSASPG		ATGTCAACTGGATTAAGGAAAA
  	STSSTAESPGPGST		AACAAAGCTCACTGGGGTGGTT
  	SSTAESPGPGSTSS		CTACCAGCGAATCTCCTTCTGG
  	TAESPGPGSTSSTA		CACCGCTCCAGGTACCTCTCCT
  	ESPGPGTSPSGESS		AGCGGCGAATCTTCTACCGCTC
  	TAPGSTSESPSGTA		CAGGTTCTACTAGCGAATCTCC
  	PGSTSESPSGTAPG		TTCTGGCACTGCACCAGGTTCT
  	TSTPESGPXXXGA		ACTAGCGAATCCCCGTCTGGTA
  	SASGAPSTXXXXS		CTGCTCCAGGTACTTCTACTCCT
  	ESPSGTAPGSTSES		GAAAGCGGTTCCGCTTCTCCAG
  	PSGTAPGSTSESPS		GTACCTCTACTCCGGAAAGCGG
  	GTAPGSTSESPSGT		TTCTGCATCTCCAGGTTCTACCA
  	APGSTSESPSGTAP		GCGAATCTCCTTCTGGCACCGC
  	GSTSESPSGTAPGT		TCCAGGTTCTACTAGCGAATCC
  	STPESGSASPGTSP		CCGTCTGGTACCGCACCAGGTA
  	SGESSTAPGTSPSG		CTTCTCCTAGCGGCGAATCTTCT
  	ESSTAPGSTSSTAE		ACCGCACCAGGTTCTACTAGCG
  	SPGPGTSPSGESST		AATCTCCGTCTGGCACTGCTCC
  	APGTSTPESGSASP		AGGTACTTCTCCTAGCGGTGAA
  	GSTSESPSGTAPGS		TCTTCTACCGCTCCAGGTACTTC
  	TSESPSGTAPGTSP		CCCTAGCGGCGAATCTTCTACC
  	SGESSTAPGSTSES		GCTCCAGGTTCTACTAGCTCTA
  	PSGTAPGTSTPESG		CTGCAGAATCTCCGGGCCCAGG
  	SASPGTSTPESGSA		TACCTCTCCTAGCGGTGAATCTT
  	SPGSTSESPSGTAP		CTACCGCTCCAGGTACTTCTCC
  	GTSTPESGSASPGS		GAGCGGTGAATCTTCTACCGCT
  	TSSTAESPGPGSTS		CCAGGTTCTACTAGCTCTACTG
  	ESPSGTAPGSTSES		CAGAATCTCCTGGCCCAGGTAC
  	PSGTAPGTSPSGES		CTCTACTCCGGAAAGCGGCTCT
  	STAPGSTSSTAESP		GCATCTCCAGGTACTTCTACCC
  	GPGTSPSGESSTAP		CTGAAAGCGGTTCTGCATCTCC
  	GTSTPESGSASPGT		AGGTTCTACTAGCGAATCTCCT
  	SPSGESSTAPGTSP		TCTGGCACTGCACCAGGTTCTA
  	SGESSTAPGTSPSG		CCAGCGAATCTCCGTCTGGCAC
  	ESSTAPGSTSSTAE		TGCACCAGGTACCTCTACCCCT
  	SPGPGSTSSTAESP		GAAAGCGGTTCCGCTTCTCCAG
  	GPGTSPSGESSTAP		GTTCTACCAGCTCTACCGCAGA
  	GSSPSASTGTGPGS		ATCTCCTGGTCCAGGTACCTCT
  	STPSGATGSPGSST		ACTCCGGAAAGCGGCTCTGCAT
  	PSGATGSP		CTCCAGGTTCTACTAGCGAATC
  			TCCTTCTGGCACTGCACCAGGT
  			ACTTCTCCGAGCGGTGAATCTT
  			CTACCGCACCAGGTTCTACTAG
  			CTCTACCGCTGAATCTCCGGGC
  			CCAGGTACTTCTCCGAGCGGTG
  			AATCTTCTACTGCTCCAGGTAC
  			CTCTACTCCTGAAAGCGGTTCT
  			GCATCTCCAGGTTCCACTAGCT
  			CTACCGCAGAATCTCCGGGCCC
  			AGGTTCTACTAGCTCTACTGCT
  			GAATCTCCTGGCCCAGGTTCTA
  			CTAGCTCTACTGCTGAATCTCC
  			GGGTCCAGGTTCTACCAGCTCT
  			ACTGCTGAATCTCCTGGTCCAG
  			GTACCTCCCCGAGCGGTGAATC
  			TTCTACTGCACCAGGTTCTACTA
  			GCGAATCTCCTTCTGGCACTGC
  			ACCAGGTTCTACCAGCGAATCT
  			CCGTCTGGCACTGCACCAGGTA
  			CCTCTACCCCTGAAAGCGGTCC
  			XXXXXXXXXXXXTGCAAGCGC
  			AAGCGGCGCGCCAAGCACGGG
  			AXXXXXXXXTAGCGAATCTCCT
  			TCTGGTACCGCTCCAGGTTCTA
  			CCAGCGAATCCCCGTCTGGTAC
  			TGCTCCAGGTTCTACCAGCGAA
  			TCTCCTTCTGGTACTGCACCAG
  			GTTCTACTAGCGAATCTCCTTCT
  			GGTACCGCTCCAGGTTCTACCA
  			GCGAATCCCCGTCTGGTACTGC
  			TCCAGGTTCTACCAGCGAATCT
  			CCTTCTGGTACTGCACCAGGTA
  			CTTCTACTCCGGAAAGCGGTTC
  			CGCATCTCCAGGTACTTCTCCTA
  			GCGGTGAATCTTCTACTGCTCC
  			AGGTACCTCTCCTAGCGGCGAA
  			TCTTCTACTGCTCCAGGTTCTAC
  			CAGCTCTACTGCTGAATCTCCG
  			GGTCCAGGTACTTCCCCGAGCG
  			GTGAATCTTCTACTGCACCAGG
  			TACTTCTACTCCGGAAAGCGGT
  			TCCGCTTCTCCAGGTTCTACCAG
  			CGAATCTCCTTCTGGCACCGCT
  			CCAGGTTCTACTAGCGAATCCC
  			CGTCTGGTACCGCACCAGGTAC
  			TTCTCCTAGCGGCGAATCTTCTA
  			CCGCACCAGGTTCTACTAGCGA
  			ATCCCCGTCTGGTACCGCACCA
  			GGTACTTCTACCCCGGAAAGCG
  			GCTCTGCTTCTCCAGGTACTTCT
  			ACCCCGGAAAGCGGCTCCGCAT
  			CTCCAGGTTCTACTAGCGAATC
  			TCCTTCTGGTACCGCTCCAGGT
  			ACTTCTACCCCTGAAAGCGGCT
  			CCGCTTCTCCAGGTTCCACTAG
  			CTCTACCGCTGAATCTCCGGGT
  			CCAGGTTCTACCAGCGAATCTC
  			CTTCTGGCACCGCTCCAGGTTCT
  			ACTAGCGAATCCCCGTCTGGTA
  			CCGCACCAGGTACTTCTCCTAG
  			CGGCGAATCTTCTACCGCACCA
  			GGTTCTACCAGCTCTACTGCTG
  			AATCTCCGGGTCCAGGTACTTC
  			CCCGAGCGGTGAATCTTCTACT
  			GCACCAGGTACTTCTACTCCGG
  			AAAGCGGTTCCGCTTCTCCAGG
  			TACCTCCCCTAGCGGCGAATCT
  			TCTACTGCTCCAGGTACCTCTCC
  			TAGCGGCGAATCTTCTACCGCT
  			CCAGGTACCTCCCCTAGCGGTG
  			AATCTTCTACCGCACCAGGTTC
  			TACTAGCTCTACTGCTGAATCTC
  			CGGGTCCAGGTTCTACCAGCTC
  			TACTGCTGAATCTCCTGGTCCA
  			GGTACCTCCCCGAGCGGTGAAT
  			CTTCTACTGCACCAGGTTCTAG
  			CCCTTCTGCTTCCACCGGTACCG
  			GCCCAGGTAGCTCTACTCCGTC
  			TGGTGCAACTGGCTCTCCAGGT
  			AGCTCTACTCCGTCTGGTGCAA
  			CCGGCTCCCCA
  FIX-	YNSGKLEEFVQGN	692	TATAATTCAGGTAAATTGGAAG	693
  AG864	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	ASPGTSSTGSPGSS		CATAATATTGAGGAGACAGAAC
  	PSASTGTGPGSSPS		ATACAGAGCAAAAGCGAAATG
  	ASTGTGPGTPGSG		TGATTCGAATTATTCCTCACCAC
  	TASSSPGSSTPSGA		AACTACAATGCAGCTATTAATA
  	TGSPGSNPSASTGT		AGTACAACCATGACATTGCCCT
  	GPGASPGTSSTGSP		TCTGGAACTGGACGAACCCTTA
  	GTPGSGTASSSPGS		GTGCTAAACAGCTACGTTACAC
  	STPSGATGSPGTPG		CTATTTGCATTGCTGACAAGGA
  	SGTASSSPGASPGT		ATACACGAACATCTTCCTCAAA
  	SSTGSPGASPGTSS		TTTGGATCTGGCTATGTAAGTG
  	TGSPGTPGSGTASS		GCTGGGGAAGAGTCTTCCACAA
  	SPGSSTPSGATGSP		AGGGAGATCAGCTTTAGTTCTT
  	GASPGTSSTGSPGT		CAGTACCTTAGAGTTCCACTTG
  	PGSGTASSSPGSST		TTGACCGAGCCACATGTCTTCG
  	PSGATGSPGSNPSA		ATCTACAAAGTTCACCATCTAT
  	STGTGPGSSPSAST		AACAACATGTTCTGTGCTGGCT
  	GTGPGSSTPSGAT		TCCATGAAGGAGGTAGAGATTC
  	GSPGSSTPSGATGS		ATGTCAAGGAGATAGTGGGGG
  	PGASPGTSSTGSPG		ACCCCATGTTACTGAAGTGGAA
  	ASPGTSSTGSPGAS		GGGACCAGTTTCTTAACTGGAA
  	PGTSSTGSPGTPGS		TTATTAGCTGGGGTGAAGAGTG
  	GTASSSPGASPGTS		TGCAATGAAAGGCAAATATGGA
  	STGSPGASPGTSST		ATATATACCAAGGTATCCCGGT
  	GSPGASPGTSSTGS		ATGTCAACTGGATTAAGGAAAA
  	PGSSPSASTGTGPG		AACAAAGCTCACTGGGGTGGTG
  	TPGSGTASSSPGAS		CTTCCCCGGGCACCAGCTCTAC
  	PGTSSTGSPGASPG		TGGTTCTCCAGGTTCTAGCCCGT
  	TSSTGSPGASPGTS		CTGCTTCTACTGGTACTGGTCCA
  	STGSPGSSTPSGAT		GGTTCTAGCCCTTCTGCTTCCAC
  	GSPGSSTPSGATGS		TGGTACTGGTCCAGGTACCCCG
  	PGASPGTSSTGSPG		GGTAGCGGTACCGCTTCTTCTTC
  	TPGSGTASSSPGSS		TCCAGGTAGCTCTACTCCGTCT
  	TPSGATGSPGSSTP		GGTGCTACCGGCTCTCCAGGTT
  	SGATGSPGSSTPSG		CTAACCCTTCTGCATCCACCGG
  	ATGSPGSSPSASTG		TACCGGCCCAGGTGCTTCTCCG
  	TGPGASPGTSSTGS		GGCACCAGCTCTACTGGTTCTC
  	PGASPGTSSTGSPG		CAGGTACCCCGGGCAGCGGTAC
  	TPGSGTASSSPGAS		CGCATCTTCTTCTCCAGGTAGCT
  	PGTSSTGSPGASPG		CTACTCCTTCTGGTGCAACTGGT
  	TSSTGSPGASPGTS		TCTCCAGGTACTCCTGGCAGCG
  	STGSPGASPGTSST		GTACCGCTTCTTCTTCTCCAGGT
  	GSPGTPGSGTASSS		GCTTCTCCTGGTACTAGCTCTAC
  	PGSSTPSGATGSPG		TGGTTCTCCAGGTGCTTCTCCGG
  	TPGSGTASSSPGSS		GCACTAGCTCTACTGGTTCTCC
  	TPSGATGSPGTPGS		AGGTACCCCGGGTAGCGGTACT
  	GTASSSPGSSTPSG		GCTTCTTCCTCTCCAGGTAGCTC
  	ATGSPGSSTPSGAT		TACCCCTTCTGGTGCAACCGGC
  	GSPGSSPSASTGTG		TCTCCAGGTGCTTCTCCGGGCA
  	PGSSPSASTGTGPG		CCAGCTCTACCGGTTCTCCAGG
  	ASPGTSSTGSPGTP		TACCCCGGGTAGCGGTACCGCT
  	GSGTASSSPGSSTP		TCTTCTTCTCCAGGTAGCTCTAC
  	SGATGSPGSSPSAS		TCCGTCTGGTGCTACCGGCTCTC
  	TGTGPGSSPSASTG		CAGGTTCTAACCCTTCTGCATCC
  	TGPGASPGTSSTGS		ACCGGTACCGGCCCAGGTTCTA
  	PGASPGTSSTGSPG		GCCCTTCTGCTTCCACCGGTACT
  	SSTPSGATGSPGSS		GGCCCAGGTAGCTCTACCCCTT
  	PSASTGTGPGASPG		CTGGTGCTACCGGCTCCCCAGG
  	TSSTGSPGSSPSAS		TAGCTCTACTCCTTCTGGTGCAA
  	TGTGPGTPGSGTA		CTGGCTCTCCAGGTGCATCTCC
  	SSSPGSSTPSGATG		GGGCACTAGCTCTACTGGTTCT
  	SPGSSTPSGATGSP		CCAGGTGCATCCCCTGGCACTA
  	GASPGTSSTGSP		GCTCTACTGGTTCTCCAGGTGCT
  			TCTCCTGGTACCAGCTCTACTG
  			GTTCTCCAGGTACTCCTGGCAG
  			CGGTACCGCTTCTTCTTCTCCAG
  			GTGCTTCTCCTGGTACTAGCTCT
  			ACTGGTTCTCCAGGTGCTTCTCC
  			GGGCACTAGCTCTACTGGTTCT
  			CCAGGTGCTTCCCCGGGCACTA
  			GCTCTACCGGTTCTCCAGGTTCT
  			AGCCCTTCTGCATCTACTGGTA
  			CTGGCCCAGGTACTCCGGGCAG
  			CGGTACTGCTTCTTCCTCTCCAG
  			GTGCATCTCCGGGCACTAGCTC
  			TACTGGTTCTCCAGGTGCATCC
  			CCTGGCACTAGCTCTACTGGTT
  			CTCCAGGTGCTTCTCCTGGTACC
  			AGCTCTACTGGTTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCAAC
  			CGGTTCCCCAGGTAGCTCTACT
  			CCTTCTGGTGCTACTGGCTCCCC
  			AGGTGCATCCCCTGGCACCAGC
  			TCTACCGGTTCTCCAGGTACCC
  			CGGGCAGCGGTACCGCATCTTC
  			CTCTCCAGGTAGCTCTACCCCG
  			TCTGGTGCTACCGGTTCCCCAG
  			GTAGCTCTACCCCGTCTGGTGC
  			AACCGGCTCCCCAGGTAGCTCT
  			ACTCCGTCTGGTGCAACCGGCT
  			CCCCAGGTTCTAGCCCGTCTGC
  			TTCCACTGGTACTGGCCCAGGT
  			GCTTCCCCGGGCACCAGCTCTA
  			CTGGTTCTCCAGGTGCATCCCC
  			GGGTACCAGCTCTACCGGTTCT
  			CCAGGTACTCCTGGCAGCGGTA
  			CTGCATCTTCCTCTCCAGGTGCT
  			TCTCCGGGCACCAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCGGG
  			CACTAGCTCTACTGGTTCTCCA
  			GGTGCATCCCCTGGCACTAGCT
  			CTACTGGTTCTCCAGGTGCTTCT
  			CCTGGTACCAGCTCTACTGGTT
  			CTCCAGGTACCCCTGGTAGCGG
  			TACTGCTTCTTCCTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCTACC
  			GGTTCTCCAGGTACCCCGGGTA
  			GCGGTACCGCATCTTCTTCTCCA
  			GGTAGCTCTACCCCGTCTGGTG
  			CTACTGGTTCTCCAGGTACTCC
  			GGGCAGCGGTACTGCTTCTTCC
  			TCTCCAGGTAGCTCTACCCCTTC
  			TGGTGCTACTGGCTCTCCAGGT
  			AGCTCTACCCCGTCTGGTGCTA
  			CTGGCTCCCCAGGTTCTAGCCC
  			TTCTGCATCCACCGGTACCGGT
  			CCAGGTTCTAGCCCGTCTGCAT
  			CTACTGGTACTGGTCCAGGTGC
  			ATCCCCGGGCACTAGCTCTACC
  			GGTTCTCCAGGTACTCCTGGTA
  			GCGGTACTGCTTCTTCTTCTCCA
  			GGTAGCTCTACTCCTTCTGGTGC
  			TACTGGTTCTCCAGGTTCTAGCC
  			CTTCTGCATCCACCGGTACCGG
  			CCCAGGTTCTAGCCCGTCTGCTT
  			CTACCGGTACTGGTCCAGGTGC
  			TTCTCCGGGTACTAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCTGGT
  			ACTAGCTCTACTGGTTCTCCAG
  			GTAGCTCTACTCCGTCTGGTGC
  			AACCGGCTCTCCAGGTTCTAGC
  			CCTTCTGCATCTACCGGTACTG
  			GTCCAGGTGCATCCCCTGGTAC
  			CAGCTCTACCGGTTCTCCAGGT
  			TCTAGCCCTTCTGCTTCTACCGG
  			TACCGGTCCAGGTACCCCTGGC
  			AGCGGTACCGCATCTTCCTCTC
  			CAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGTTCCCCAGGTAGC
  			TCTACTCCTTCTGGTGCTACTGG
  			CTCCCCAGGTGCATCCCCTGGC
  			ACCAGCTCTACCGGTTCTCCA
  FIX-	YNSGKLEEFVQGN	694	TATAATTCAGGTAAATTGGAAG	695
  AM875	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	TSTEPSEGSAPGSE		CATAATATTGAGGAGACAGAAC
  	PATSGSETPGSPAG		ATACAGAGCAAAAGCGAAATG
  	SPTSTEEGSTSSTA		TGATTCGAATTATTCCTCACCAC
  	ESPGPGTSTPESGS		AACTACAATGCAGCTATTAATA
  	ASPGSTSESPSGTA		AGTACAACCATGACATTGCCCT
  	PGSTSESPSGTAPG		TCTGGAACTGGACGAACCCTTA
  	TSTPESGSASPGTS		GTGCTAAACAGCTACGTTACAC
  	TPESGSASPGSEPA		CTATTTGCATTGCTGACAAGGA
  	TSGSETPGTSESAT		ATACACGAACATCTTCCTCAAA
  	PESGPGSPAGSPTS		TTTGGATCTGGCTATGTAAGTG
  	TEEGTSTEPSEGSA		GCTGGGGAAGAGTCTTCCACAA
  	PGTSESATPESGPG		AGGGAGATCAGCTTTAGTTCTT
  	TSTEPSEGSAPGTS		CAGTACCTTAGAGTTCCACTTG
  	TEPSEGSAPGSPAG		TTGACCGAGCCACATGTCTTCG
  	SPTSTEEGTSTEPS		ATCTACAAAGTTCACCATCTAT
  	EGSAPGTSTEPSEG		AACAACATGTTCTGTGCTGGCT
  	SAPGTSESATPESG		TCCATGAAGGAGGTAGAGATTC
  	PGTSESATPESGPG		ATGTCAAGGAGATAGTGGGGG
  	TSTEPSEGSAPGTS		ACCCCATGTTACTGAAGTGGAA
  	TEPSEGSAPGTSES		GGGACCAGTTTCTTAACTGGAA
  	ATPESGPGTSTEPS		TTATTAGCTGGGGTGAAGAGTG
  	EGSAPGSEPATSGS		TGCAATGAAAGGCAAATATGGA
  	ETPGSPAGSPTSTE		ATATATACCAAGGTATCCCGGT
  	EGSSTPSGATGSPG		ATGTCAACTGGATTAAGGAAAA
  	TPGSGTASSSPGSS		AACAAAGCTCACTGGGGTGGTA
  	TPSGATGSPGTSTE		CTTCTACTGAACCGTCTGAAGG
  	PSEGSAPGTSTEPS		CAGCGCACCAGGTAGCGAACCG
  	EGSAPGSEPATSGS		GCTACTTCCGGTTCTGAAACCC
  	ETPGSPAGSPTSTE		CAGGTAGCCCAGCAGGTTCTCC
  	EGSPAGSPTSTEEG		AACTTCTACTGAAGAAGGTTCT
  	TSTEPSEGSAPGAS		ACCAGCTCTACCGCAGAATCTC
  	ASGAPSTGGTSES		CTGGTCCAGGTACCTCTACTCC
  	ATPESGPGSPAGSP		GGAAAGCGGCTCTGCATCTCCA
  	TSTEEGSPAGSPTS		GGTTCTACTAGCGAATCTCCTTC
  	TEEGSTSSTAESPG		TGGCACTGCACCAGGTTCTACT
  	PGSTSESPSGTAPG		AGCGAATCCCCGTCTGGTACTG
  	TSPSGESSTAPGTP		CTCCAGGTACTTCTACTCCTGA
  	GSGTASSSPGSSTP		AAGCGGTTCCGCTTCTCCAGGT
  	SGATGSPGSSPSAS		ACCTCTACTCCGGAAAGCGGTT
  	TGTGPGSEPATSGS		CTGCATCTCCAGGTAGCGAACC
  	ETPGTSESATPESG		GGCAACCTCCGGCTCTGAAACC
  	PGSEPATSGSETPG		CCAGGTACCTCTGAAAGCGCTA
  	STSSTAESPGPGST		CTCCTGAATCCGGCCCAGGTAG
  	SSTAESPGPGTSPS		CCCGGCAGGTTCTCCGACTTCC
  	GESSTAPGSEPATS		ACTGAGGAAGGTACCTCTACTG
  	GSETPGSEPATSGS		AACCTTCTGAGGGCAGCGCTCC
  	ETPGTSTEPSEGSA		AGGTACTTCTGAAAGCGCTACC
  	PGSTSSTAESPGPG		CCGGAGTCCGGTCCAGGTACTT
  	TSTPESGSASPGST		CTACTGAACCGTCCGAAGGTAG
  	SESPSGTAPGTSTE		CGCACCAGGTACTTCTACCGAA
  	PSEGSAPGTSTEPS		CCGTCCGAGGGTAGCGCACCAG
  	EGSAPGTSTEPSEG		GTAGCCCAGCAGGTTCTCCTAC
  	SAPGSSTPSGATGS		CTCCACCGAGGAAGGTACTTCT
  	PGSSPSASTGTGPG		ACCGAACCGTCCGAGGGTAGCG
  	ASPGTSSTGSPGSE		CACCAGGTACTTCTACCGAACC
  	PATSGSETPGTSES		TTCCGAGGGCAGCGCACCAGGT
  	ATPESGPGSPAGSP		ACTTCTGAAAGCGCTACCCCTG
  	TSTEEGSSTPSGAT		AGTCCGGCCCAGGTACTTCTGA
  	GSPGSSPSASTGTG		AAGCGCTACTCCTGAATCCGGT
  	PGASPGTSSTGSPG		CCAGGTACCTCTACTGAACCTT
  	TSESATPESGPGTS		CCGAAGGCAGCGCTCCAGGTAC
  	TEPSEGSAPGTSTE		CTCTACCGAACCGTCCGAGGGC
  	PSEGSAP		AGCGCACCAGGTACTTCTGAAA
  			GCGCAACCCCTGAATCCGGTCC
  			AGGTACTTCTACTGAACCTTCC
  			GAAGGTAGCGCTCCAGGTAGCG
  			AACCTGCTACTTCTGGTTCTGA
  			AACCCCAGGTAGCCCGGCTGGC
  			TCTCCGACCTCCACCGAGGAAG
  			GTAGCTCTACCCCGTCTGGTGC
  			TACTGGTTCTCCAGGTACTCCG
  			GGCAGCGGTACTGCTTCTTCCT
  			CTCCAGGTAGCTCTACCCCTTCT
  			GGTGCTACTGGCTCTCCAGGTA
  			CCTCTACCGAACCGTCCGAGGG
  			TAGCGCACCAGGTACCTCTACT
  			GAACCGTCTGAGGGTAGCGCTC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACTCCAGGTAGC
  			CCTGCTGGCTCTCCGACTTCTAC
  			TGAGGAAGGTAGCCCGGCTGGT
  			TCTCCGACTTCTACTGAGGAAG
  			GTACTTCTACCGAACCTTCCGA
  			AGGTAGCGCTCCAGGTGCAAGC
  			GCAAGCGGCGCGCCAAGCACG
  			GGAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTTCTACCAGCTCTACCGCT
  			GAATCTCCTGGCCCAGGTTCTA
  			CTAGCGAATCTCCGTCTGGCAC
  			CGCACCAGGTACTTCCCCTAGC
  			GGTGAATCTTCTACTGCACCAG
  			GTACCCCTGGCAGCGGTACCGC
  			TTCTTCCTCTCCAGGTAGCTCTA
  			CCCCGTCTGGTGCTACTGGCTCT
  			CCAGGTTCTAGCCCGTCTGCAT
  			CTACCGGTACCGGCCCAGGTAG
  			CGAACCGGCAACCTCCGGCTCT
  			GAAACTCCAGGTACTTCTGAAA
  			GCGCTACTCCGGAATCCGGCCC
  			AGGTAGCGAACCGGCTACTTCC
  			GGCTCTGAAACCCCAGGTTCCA
  			CCAGCTCTACTGCAGAATCTCC
  			GGGCCCAGGTTCTACTAGCTCT
  			ACTGCAGAATCTCCGGGTCCAG
  			GTACTTCTCCTAGCGGCGAATC
  			TTCTACCGCTCCAGGTAGCGAA
  			CCGGCAACCTCTGGCTCTGAAA
  			CTCCAGGTAGCGAACCTGCAAC
  			CTCCGGCTCTGAAACCCCAGGT
  			ACTTCTACTGAACCTTCTGAGG
  			GCAGCGCACCAGGTTCTACCAG
  			CTCTACCGCAGAATCTCCTGGT
  			CCAGGTACCTCTACTCCGGAAA
  			GCGGCTCTGCATCTCCAGGTTC
  			TACTAGCGAATCTCCTTCTGGC
  			ACTGCACCAGGTACTTCTACCG
  			AACCGTCCGAAGGCAGCGCTCC
  			AGGTACCTCTACTGAACCTTCC
  			GAGGGCAGCGCTCCAGGTACCT
  			CTACCGAACCTTCTGAAGGTAG
  			CGCACCAGGTAGCTCTACTCCG
  			TCTGGTGCAACCGGCTCCCCAG
  			GTTCTAGCCCGTCTGCTTCCACT
  			GGTACTGGCCCAGGTGCTTCCC
  			CGGGCACCAGCTCTACTGGTTC
  			TCCAGGTAGCGAACCTGCTACC
  			TCCGGTTCTGAAACCCCAGGTA
  			CCTCTGAAAGCGCAACTCCGGA
  			GTCTGGTCCAGGTAGCCCTGCA
  			GGTTCTCCTACCTCCACTGAGG
  			AAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGCTCCCCAGGTTCT
  			AGCCCGTCTGCTTCCACTGGTA
  			CTGGCCCAGGTGCTTCCCCGGG
  			CACCAGCTCTACTGGTTCTCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CGGAGTCTGGCCCAGGTACCTC
  			TACTGAACCGTCTGAGGGTAGC
  			GCTCCAGGTACTTCTACTGAAC
  			CGTCCGAAGGTAGCGCACCA
  FIX-	YNSGKLEEFVQGN	696	TATAATTCAGGTAAATTGGAAG	697
  AG864	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	ASPGTSSTGSPGSS		CATAATATTGAGGAGACAGAAC
  	PSASTGTGPGSSPS		ATACAGAGCAAAAGCGAAATG
  	ASTGTGPGTPGSG		TGATTCGAATTATTCCTCACCAC
  	TASSSPGSSTPSGA		AACTACAATGCAGCTATTAATA
  	TGSPGSNPSASTGT		AGTACAACCATGACATTGCCCT
  	GPGASPGTSSTGSP		TCTGGAACTGGACGAACCCTTA
  	GTPGSGTASSSPGS		GTGCTAAACAGCTACGTTACAC
  	STPSGATGSPGTPG		CTATTTGCATTGCTGACAAGGA
  	SGTASSSPGASPGT		ATACACGAACATCTTCCTCAAA
  	SSTGSPGASPGTSS		TTTGGATCTGGCTATGTAAGTG
  	TGSPGTPGSGTASS		GCTGGGGAAGAGTCTTCCACAA
  	SPGSSTPSGATGSP		AGGGAGATCAGCTTTAGTTCTT
  	GASPGTSSTGSPGT		CAGTACCTTAGAGTTCCACTTG
  	PGSGTASSSPGSST		TTGACCGAGCCACATGTCTTCG
  	PSGATGSPGSNPSA		ATCTACAAAGTTCACCATCTAT
  	STGTGPGSSPSAST		AACAACATGTTCTGTGCTGGCT
  	GTGPGSSTPSGAT		TCCATGAAGGAGGTAGAGATTC
  	GSPGSSTPSGATGS		ATGTCAAGGAGATAGTGGGGG
  	PGASPGTSSTGSPG		ACCCCATGTTACTGAAGTGGAA
  	ASPGTSSTGSPGAS		GGGACCAGTTTCTTAACTGGAA
  	PGTSSTGSPGTPGS		TTATTAGCTGGGGTGAAGAGTG
  	GTASSSPGASPGTS		TGCAATGAAAGGCAAATATGGA
  	STGSPGASPGTSST		ATATATACCAAGGTATCCCGGT
  	GSPGASPGTSSTGS		ATGTCAACTGGATTAAGGAAAA
  	PGSSPSASTGTGPG		AACAAAGCTCACTGGGGTGGTG
  	TPGSGTASSSPGAS		CTTCCCCGGGCACCAGCTCTAC
  	PGTSSTGSPGASPG		TGGTTCTCCAGGTTCTAGCCCGT
  	TSSTGSPGASPGTS		CTGCTTCTACTGGTACTGGTCCA
  	STGSPGSSTPSGAT		GGTTCTAGCCCTTCTGCTTCCAC
  	GSPGSSTPSGATGS		TGGTACTGGTCCAGGTACCCCG
  	PGASPGTSSTGSPG		GGTAGCGGTACCGCTTCTTCTTC
  	TPGSGTASSSPGSS		TCCAGGTAGCTCTACTCCGTCT
  	TPSGATGSPGSSTP		GGTGCTACCGGCTCTCCAGGTT
  	SGATGSPGSSTPSG		CTAACCCTTCTGCATCCACCGG
  	ATGSPGSSPSASTG		TACCGGCCCAGGTGCTTCTCCG
  	TGPGASPGTSSTGS		GGCACCAGCTCTACTGGTTCTC
  	PGASPGTSSTGSPG		CAGGTACCCCGGGCAGCGGTAC
  	TPGSGTASSSPGAS		CGCATCTTCTTCTCCAGGTAGCT
  	PGTSSTGSPGASPG		CTACTCCTTCTGGTGCAACTGGT
  	TSSTGSPGASPGTS		TCTCCAGGTACTCCTGGCAGCG
  	STGSPGASPGTSST		GTACCGCTTCTTCTTCTCCAGGT
  	GSPGTPGSGTASSS		GCTTCTCCTGGTACTAGCTCTAC
  	PGSSTPSGATGSPG		TGGTTCTCCAGGTGCTTCTCCGG
  	TPGSGTASSSPGSS		GCACTAGCTCTACTGGTTCTCC
  	TPSGATGSPGTPGS		AGGTACCCCGGGTAGCGGTACT
  	GTASSSPGSSTPSG		GCTTCTTCCTCTCCAGGTAGCTC
  	ATGSPGSSTPSGAT		TACCCCTTCTGGTGCAACCGGC
  	GSPGSSPSASTGTG		TCTCCAGGTGCTTCTCCGGGCA
  	PGSSPSASTGTGPG		CCAGCTCTACCGGTTCTCCAGG
  	ASPGTSSTGSPGTP		TACCCCGGGTAGCGGTACCGCT
  	GSGTASSSPGSSTP		TCTTCTTCTCCAGGTAGCTCTAC
  	SGATGSPGSSPSAS		TCCGTCTGGTGCTACCGGCTCTC
  	TGTGPGSSPSASTG		CAGGTTCTAACCCTTCTGCATCC
  	TGPGASPGTSSTGS		ACCGGTACCGGCCCAGGTTCTA
  	PGASPGTSSTGSPG		GCCCTTCTGCTTCCACCGGTACT
  	SSTPSGATGSPGSS		GGCCCAGGTAGCTCTACCCCTT
  	PSASTGTGPGASPG		CTGGTGCTACCGGCTCCCCAGG
  	TSSTGSPGSSPSAS		TAGCTCTACTCCTTCTGGTGCAA
  	TGTGPGTPGSGTA		CTGGCTCTCCAGGTGCATCTCC
  	SSSPGSSTPSGATG		GGGCACTAGCTCTACTGGTTCT
  	SPGSSTPSGATGSP		CCAGGTGCATCCCCTGGCACTA
  	GASPGTSSTGSP		GCTCTACTGGTTCTCCAGGTGCT
  			TCTCCTGGTACCAGCTCTACTG
  			GTTCTCCAGGTACTCCTGGCAG
  			CGGTACCGCTTCTTCTTCTCCAG
  			GTGCTTCTCCTGGTACTAGCTCT
  			ACTGGTTCTCCAGGTGCTTCTCC
  			GGGCACTAGCTCTACTGGTTCT
  			CCAGGTGCTTCCCCGGGCACTA
  			GCTCTACCGGTTCTCCAGGTTCT
  			AGCCCTTCTGCATCTACTGGTA
  			CTGGCCCAGGTACTCCGGGCAG
  			CGGTACTGCTTCTTCCTCTCCAG
  			GTGCATCTCCGGGCACTAGCTC
  			TACTGGTTCTCCAGGTGCATCC
  			CCTGGCACTAGCTCTACTGGTT
  			CTCCAGGTGCTTCTCCTGGTACC
  			AGCTCTACTGGTTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCAAC
  			CGGTTCCCCAGGTAGCTCTACT
  			CCTTCTGGTGCTACTGGCTCCCC
  			AGGTGCATCCCCTGGCACCAGC
  			TCTACCGGTTCTCCAGGTACCC
  			CGGGCAGCGGTACCGCATCTTC
  			CTCTCCAGGTAGCTCTACCCCG
  			TCTGGTGCTACCGGTTCCCCAG
  			GTAGCTCTACCCCGTCTGGTGC
  			AACCGGCTCCCCAGGTAGCTCT
  			ACTCCGTCTGGTGCAACCGGCT
  			CCCCAGGTTCTAGCCCGTCTGC
  			TTCCACTGGTACTGGCCCAGGT
  			GCTTCCCCGGGCACCAGCTCTA
  			CTGGTTCTCCAGGTGCATCCCC
  			GGGTACCAGCTCTACCGGTTCT
  			CCAGGTACTCCTGGCAGCGGTA
  			CTGCATCTTCCTCTCCAGGTGCT
  			TCTCCGGGCACCAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCGGG
  			CACTAGCTCTACTGGTTCTCCA
  			GGTGCATCCCCTGGCACTAGCT
  			CTACTGGTTCTCCAGGTGCTTCT
  			CCTGGTACCAGCTCTACTGGTT
  			CTCCAGGTACCCCTGGTAGCGG
  			TACTGCTTCTTCCTCTCCAGGTA
  			GCTCTACTCCGTCTGGTGCTACC
  			GGTTCTCCAGGTACCCCGGGTA
  			GCGGTACCGCATCTTCTTCTCCA
  			GGTAGCTCTACCCCGTCTGGTG
  			CTACTGGTTCTCCAGGTACTCC
  			GGGCAGCGGTACTGCTTCTTCC
  			TCTCCAGGTAGCTCTACCCCTTC
  			TGGTGCTACTGGCTCTCCAGGT
  			AGCTCTACCCCGTCTGGTGCTA
  			CTGGCTCCCCAGGTTCTAGCCC
  			TTCTGCATCCACCGGTACCGGT
  			CCAGGTTCTAGCCCGTCTGCAT
  			CTACTGGTACTGGTCCAGGTGC
  			ATCCCCGGGCACTAGCTCTACC
  			GGTTCTCCAGGTACTCCTGGTA
  			GCGGTACTGCTTCTTCTTCTCCA
  			GGTAGCTCTACTCCTTCTGGTGC
  			TACTGGTTCTCCAGGTTCTAGCC
  			CTTCTGCATCCACCGGTACCGG
  			CCCAGGTTCTAGCCCGTCTGCTT
  			CTACCGGTACTGGTCCAGGTGC
  			TTCTCCGGGTACTAGCTCTACTG
  			GTTCTCCAGGTGCATCTCCTGGT
  			ACTAGCTCTACTGGTTCTCCAG
  			GTAGCTCTACTCCGTCTGGTGC
  			AACCGGCTCTCCAGGTTCTAGC
  			CCTTCTGCATCTACCGGTACTG
  			GTCCAGGTGCATCCCCTGGTAC
  			CAGCTCTACCGGTTCTCCAGGT
  			TCTAGCCCTTCTGCTTCTACCGG
  			TACCGGTCCAGGTACCCCTGGC
  			AGCGGTACCGCATCTTCCTCTC
  			CAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGTTCCCCAGGTAGC
  			TCTACTCCTTCTGGTGCTACTGG
  			CTCCCCAGGTGCATCCCCTGGC
  			ACCAGCTCTACCGGTTCTCCA
  FIX-	YNSGKLEEFVQGN	698	TATAATTCAGGTAAATTGGAAG	699
  AM875	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	TSTEPSEGSAPGSE		CATAATATTGAGGAGACAGAAC
  	PATSGSETPGSPAG		ATACAGAGCAAAAGCGAAATG
  	SPTSTEEGSTSSTA		TGATTCGAATTATTCCTCACCAC
  	ESPGPGTSTPESGS		AACTACAATGCAGCTATTAATA
  	ASPGSTSESPSGTA		AGTACAACCATGACATTGCCCT
  	PGSTSESPSGTAPG		TCTGGAACTGGACGAACCCTTA
  	TSTPESGSASPGTS		GTGCTAAACAGCTACGTTACAC
  	TPESGSASPGSEPA		CTATTTGCATTGCTGACAAGGA
  	TSGSETPGTSESAT		ATACACGAACATCTTCCTCAAA
  	PESGPGSPAGSPTS		TTTGGATCTGGCTATGTAAGTG
  	TEEGTSTEPSEGSA		GCTGGGGAAGAGTCTTCCACAA
  	PGTSESATPESGPG		AGGGAGATCAGCTTTAGTTCTT
  	TSTEPSEGSAPGTS		CAGTACCTTAGAGTTCCACTTG
  	TEPSEGSAPGSPAG		TTGACCGAGCCACATGTCTTCG
  	SPTSTEEGTSTEPS		ATCTACAAAGTTCACCATCTAT
  	EGSAPGTSTEPSEG		AACAACATGTTCTGTGCTGGCT
  	SAPGTSESATPESG		TCCATGAAGGAGGTAGAGATTC
  	PGTSESATPESGPG		ATGTCAAGGAGATAGTGGGGG
  	TSTEPSEGSAPGTS		ACCCCATGTTACTGAAGTGGAA
  	TEPSEGSAPGTSES		GGGACCAGTTTCTTAACTGGAA
  	ATPESGPGTSTEPS		TTATTAGCTGGGGTGAAGAGTG
  	EGSAPGSEPATSGS		TGCAATGAAAGGCAAATATGGA
  	ETPGSPAGSPTSTE		ATATATACCAAGGTATCCCGGT
  	EGSSTPSGATGSPG		ATGTCAACTGGATTAAGGAAAA
  	TPGSGTASSSPGSS		AACAAAGCTCACTGGGGTGGTA
  	TPSGATGSPGTSTE		CTTCTACTGAACCGTCTGAAGG
  	PSEGSAPGTSTEPS		CAGCGCACCAGGTAGCGAACCG
  	EGSAPGSEPATSGS		GCTACTTCCGGTTCTGAAACCC
  	ETPGSPAGSPTSTE		CAGGTAGCCCAGCAGGTTCTCC
  	EGSPAGSPTSTEEG		AACTTCTACTGAAGAAGGTTCT
  	TSTEPSEGSAPGAS		ACCAGCTCTACCGCAGAATCTC
  	ASGAPSTGGTSES		CTGGTCCAGGTACCTCTACTCC
  	ATPESGPGSPAGSP		GGAAAGCGGCTCTGCATCTCCA
  	TSTEEGSPAGSPTS		GGTTCTACTAGCGAATCTCCTTC
  	TEEGSTSSTAESPG		TGGCACTGCACCAGGTTCTACT
  	PGSTSESPSGTAPG		AGCGAATCCCCGTCTGGTACTG
  	TSPSGESSTAPGTP		CTCCAGGTACTTCTACTCCTGA
  	GSGTASSSPGSSTP		AAGCGGTTCCGCTTCTCCAGGT
  	SGATGSPGSSPSAS		ACCTCTACTCCGGAAAGCGGTT
  	TGTGPGSEPATSGS		CTGCATCTCCAGGTAGCGAACC
  	ETPGTSESATPESG		GGCAACCTCCGGCTCTGAAACC
  	PGSEPATSGSETPG		CCAGGTACCTCTGAAAGCGCTA
  	STSSTAESPGPGST		CTCCTGAATCCGGCCCAGGTAG
  	SSTAESPGPGTSPS		CCCGGCAGGTTCTCCGACTTCC
  	GESSTAPGSEPATS		ACTGAGGAAGGTACCTCTACTG
  	GSETPGSEPATSGS		AACCTTCTGAGGGCAGCGCTCC
  	ETPGTSTEPSEGSA		AGGTACTTCTGAAAGCGCTACC
  	PGSTSSTAESPGPG		CCGGAGTCCGGTCCAGGTACTT
  	TSTPESGSASPGST		CTACTGAACCGTCCGAAGGTAG
  	SESPSGTAPGTSTE		CGCACCAGGTACTTCTACCGAA
  	PSEGSAPGTSTEPS		CCGTCCGAGGGTAGCGCACCAG
  	EGSAPGTSTEPSEG		GTAGCCCAGCAGGTTCTCCTAC
  	SAPGSSTPSGATGS		CTCCACCGAGGAAGGTACTTCT
  	PGSSPSASTGTGPG		ACCGAACCGTCCGAGGGTAGCG
  	ASPGTSSTGSPGSE		CACCAGGTACTTCTACCGAACC
  	PATSGSETPGTSES		TTCCGAGGGCAGCGCACCAGGT
  	ATPESGPGSPAGSP		ACTTCTGAAAGCGCTACCCCTG
  	TSTEEGSSTPSGAT		AGTCCGGCCCAGGTACTTCTGA
  	GSPGSSPSASTGTG		AAGCGCTACTCCTGAATCCGGT
  	PGASPGTSSTGSPG		CCAGGTACCTCTACTGAACCTT
  	TSESATPESGPGTS		CCGAAGGCAGCGCTCCAGGTAC
  	TEPSEGSAPGTSTE		CTCTACCGAACCGTCCGAGGGC
  	PSEGSAP		AGCGCACCAGGTACTTCTGAAA
  			GCGCAACCCCTGAATCCGGTCC
  			AGGTACTTCTACTGAACCTTCC
  			GAAGGTAGCGCTCCAGGTAGCG
  			AACCTGCTACTTCTGGTTCTGA
  			AACCCCAGGTAGCCCGGCTGGC
  			TCTCCGACCTCCACCGAGGAAG
  			GTAGCTCTACCCCGTCTGGTGC
  			TACTGGTTCTCCAGGTACTCCG
  			GGCAGCGGTACTGCTTCTTCCT
  			CTCCAGGTAGCTCTACCCCTTCT
  			GGTGCTACTGGCTCTCCAGGTA
  			CCTCTACCGAACCGTCCGAGGG
  			TAGCGCACCAGGTACCTCTACT
  			GAACCGTCTGAGGGTAGCGCTC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACTCCAGGTAGC
  			CCTGCTGGCTCTCCGACTTCTAC
  			TGAGGAAGGTAGCCCGGCTGGT
  			TCTCCGACTTCTACTGAGGAAG
  			GTACTTCTACCGAACCTTCCGA
  			AGGTAGCGCTCCAGGTGCAAGC
  			GCAAGCGGCGCGCCAAGCACG
  			GGAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTTCTACCAGCTCTACCGCT
  			GAATCTCCTGGCCCAGGTTCTA
  			CTAGCGAATCTCCGTCTGGCAC
  			CGCACCAGGTACTTCCCCTAGC
  			GGTGAATCTTCTACTGCACCAG
  			GTACCCCTGGCAGCGGTACCGC
  			TTCTTCCTCTCCAGGTAGCTCTA
  			CCCCGTCTGGTGCTACTGGCTCT
  			CCAGGTTCTAGCCCGTCTGCAT
  			CTACCGGTACCGGCCCAGGTAG
  			CGAACCGGCAACCTCCGGCTCT
  			GAAACTCCAGGTACTTCTGAAA
  			GCGCTACTCCGGAATCCGGCCC
  			AGGTAGCGAACCGGCTACTTCC
  			GGCTCTGAAACCCCAGGTTCCA
  			CCAGCTCTACTGCAGAATCTCC
  			GGGCCCAGGTTCTACTAGCTCT
  			ACTGCAGAATCTCCGGGTCCAG
  			GTACTTCTCCTAGCGGCGAATC
  			TTCTACCGCTCCAGGTAGCGAA
  			CCGGCAACCTCTGGCTCTGAAA
  			CTCCAGGTAGCGAACCTGCAAC
  			CTCCGGCTCTGAAACCCCAGGT
  			ACTTCTACTGAACCTTCTGAGG
  			GCAGCGCACCAGGTTCTACCAG
  			CTCTACCGCAGAATCTCCTGGT
  			CCAGGTACCTCTACTCCGGAAA
  			GCGGCTCTGCATCTCCAGGTTC
  			TACTAGCGAATCTCCTTCTGGC
  			ACTGCACCAGGTACTTCTACCG
  			AACCGTCCGAAGGCAGCGCTCC
  			AGGTACCTCTACTGAACCTTCC
  			GAGGGCAGCGCTCCAGGTACCT
  			CTACCGAACCTTCTGAAGGTAG
  			CGCACCAGGTAGCTCTACTCCG
  			TCTGGTGCAACCGGCTCCCCAG
  			GTTCTAGCCCGTCTGCTTCCACT
  			GGTACTGGCCCAGGTGCTTCCC
  			CGGGCACCAGCTCTACTGGTTC
  			TCCAGGTAGCGAACCTGCTACC
  			TCCGGTTCTGAAACCCCAGGTA
  			CCTCTGAAAGCGCAACTCCGGA
  			GTCTGGTCCAGGTAGCCCTGCA
  			GGTTCTCCTACCTCCACTGAGG
  			AAGGTAGCTCTACTCCGTCTGG
  			TGCAACCGGCTCCCCAGGTTCT
  			AGCCCGTCTGCTTCCACTGGTA
  			CTGGCCCAGGTGCTTCCCCGGG
  			CACCAGCTCTACTGGTTCTCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CGGAGTCTGGCCCAGGTACCTC
  			TACTGAACCGTCTGAGGGTAGC
  			GCTCCAGGTACTTCTACTGAAC
  			CGTCCGAAGGTAGCGCACCA
  FIX-	YNSGKLEEFVQGN	700	TATAATTCAGGTAAATTGGAAG	701
  AM1318	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGG		AATTACAGTTGTCGCAGGTGAA
  	TSTEPSEGSAPGSE		CATAATATTGAGGAGACAGAAC
  	PATSGSETPGSPAG		ATACAGAGCAAAAGCGAAATG
  	SPTSTEEGSTSSTA		TGATTCGAATTATTCCTCACCAC
  	ESPGPGTSTPESGS		AACTACAATGCAGCTATTAATA
  	ASPGSTSESPSGTA		AGTACAACCATGACATTGCCCT
  	PGSTSESPSGTAPG		TCTGGAACTGGACGAACCCTTA
  	TSTPESGSASPGTS		GTGCTAAACAGCTACGTTACAC
  	TPESGSASPGSEPA		CTATTTGCATTGCTGACAAGGA
  	TSGSETPGTSESAT		ATACACGAACATCTTCCTCAAA
  	PESGPGSPAGSPTS		TTTGGATCTGGCTATGTAAGTG
  	TEEGTSTEPSEGSA		GCTGGGGAAGAGTCTTCCACAA
  	PGTSESATPESGPG		AGGGAGATCAGCTTTAGTTCTT
  	TSTEPSEGSAPGTS		CAGTACCTTAGAGTTCCACTTG
  	TEPSEGSAPGSPAG		TTGACCGAGCCACATGTCTTCG
  	SPTSTEEGTSTEPS		ATCTACAAAGTTCACCATCTAT
  	EGSAPGTSTEPSEG		AACAACATGTTCTGTGCTGGCT
  	SAPGTSESATPESG		TCCATGAAGGAGGTAGAGATTC
  	PGTSESATPESGPG		ATGTCAAGGAGATAGTGGGGG
  	TSTEPSEGSAPGTS		ACCCCATGTTACTGAAGTGGAA
  	TEPSEGSAPGTSES		GGGACCAGTTTCTTAACTGGAA
  	ATPESGPGTSTEPS		TTATTAGCTGGGGTGAAGAGTG
  	EGSAPGSEPATSGS		TGCAATGAAAGGCAAATATGGA
  	ETPGSPAGSPTSTE		ATATATACCAAGGTATCCCGGT
  	EGSSTPSGATGSPG		ATGTCAACTGGATTAAGGAAAA
  	TPGSGTASSSPGSS		AACAAAGCTCACTGGGGTGGTA
  	TPSGATGSPGTSTE		CTTCTACTGAACCGTCTGAAGG
  	PSEGSAPGTSTEPS		CAGCGCACCAGGTAGCGAACCG
  	EGSAPGSEPATSGS		GCTACTTCCGGTTCTGAAACCC
  	ETPGSPAGSPTSTE		CAGGTAGCCCAGCAGGTTCTCC
  	EGSPAGSPTSTEEG		AACTTCTACTGAAGAAGGTTCT
  	TSTEPSEGSAPGPE		ACCAGCTCTACCGCAGAATCTC
  	PTGPAPSGGSEPAT		CTGGTCCAGGTACCTCTACTCC
  	SGSETPGTSESATP		GGAAAGCGGCTCTGCATCTCCA
  	ESGPGSPAGSPTST		GGTTCTACTAGCGAATCTCCTTC
  	EEGTSESATPESGP		TGGCACTGCACCAGGTTCTACT
  	GSPAGSPTSTEEGS		AGCGAATCCCCGTCTGGTACTG
  	PAGSPTSTEEGTSE		CTCCAGGTACTTCTACTCCTGA
  	SATPESGPGSPAGS		AAGCGGTTCCGCTTCTCCAGGT
  	PTSTEEGSPAGSPT		ACCTCTACTCCGGAAAGCGGTT
  	STEEGSTSSTAESP		CTGCATCTCCAGGTAGCGAACC
  	GPGSTSESPSGTAP		GGCAACCTCCGGCTCTGAAACC
  	GTSPSGESSTAPGS		CCAGGTACCTCTGAAAGCGCTA
  	TSESPSGTAPGSTS		CTCCTGAATCCGGCCCAGGTAG
  	ESPSGTAPGTSPSG		CCCGGCAGGTTCTCCGACTTCC
  	ESSTAPGTSTEPSE		ACTGAGGAAGGTACCTCTACTG
  	GSAPGTSESATPES		AACCTTCTGAGGGCAGCGCTCC
  	GPGTSESATPESGP		AGGTACTTCTGAAAGCGCTACC
  	GSEPATSGSETPGT		CCGGAGTCCGGTCCAGGTACTT
  	SESATPESGPGTSE		CTACTGAACCGTCCGAAGGTAG
  	SATPESGPGTSTEP		CGCACCAGGTACTTCTACCGAA
  	SEGSAPGTSESATP		CCGTCCGAGGGTAGCGCACCAG
  	ESGPGTSTEPSEGS		GTAGCCCAGCAGGTTCTCCTAC
  	APGTSPSGESSTAP		CTCCACCGAGGAAGGTACTTCT
  	GTSPSGESSTAPGT		ACCGAACCGTCCGAGGGTAGCG
  	SPSGESSTAPGTST		CACCAGGTACTTCTACCGAACC
  	EPSEGSAPGSPAGS		TTCCGAGGGCAGCGCACCAGGT
  	PTSTEEGTSTEPSE		ACTTCTGAAAGCGCTACCCCTG
  	GSAPGSSPSASTGT		AGTCCGGCCCAGGTACTTCTGA
  	GPGSSTPSGATGSP		AAGCGCTACTCCTGAATCCGGT
  	GSSTPSGATGSPGS		CCAGGTACCTCTACTGAACCTT
  	STPSGATGSPGSST		CCGAAGGCAGCGCTCCAGGTAC
  	PSGATGSPGASPGT		CTCTACCGAACCGTCCGAGGGC
  	SSTGSPGASASGAP		AGCGCACCAGGTACTTCTGAAA
  	STGGTSPSGESSTA		GCGCAACCCCTGAATCCGGTCC
  	PGSTSSTAESPGPG		AGGTACTTCTACTGAACCTTCC
  	TSPSGESSTAPGTS		GAAGGTAGCGCTCCAGGTAGCG
  	ESATPESGPGTSTE		AACCTGCTACTTCTGGTTCTGA
  	PSEGSAPGTSTEPS		AACCCCAGGTAGCCCGGCTGGC
  	EGSAPGSSPSASTG		TCTCCGACCTCCACCGAGGAAG
  	TGPGSSTPSGATGS		GTAGCTCTACCCCGTCTGGTGC
  	PGASPGTSSTGSPG		TACTGGTTCTCCAGGTACTCCG
  	TSTPESGSASPGTS		GGCAGCGGTACTGCTTCTTCCT
  	PSGESSTAPGTSPS		CTCCAGGTAGCTCTACCCCTTCT
  	GESSTAPGTSESAT		GGTGCTACTGGCTCTCCAGGTA
  	PESGPGSEPATSGS		CCTCTACCGAACCGTCCGAGGG
  	ETPGTSTEPSEGSA		TAGCGCACCAGGTACCTCTACT
  	PGSTSESPSGTAPG		GAACCGTCTGAGGGTAGCGCTC
  	STSESPSGTAPGTS		CAGGTAGCGAACCGGCAACCTC
  	TPESGSASPGSPAG		CGGTTCTGAAACTCCAGGTAGC
  	SPTSTEEGTSESAT		CCTGCTGGCTCTCCGACTTCTAC
  	PESGPGTSTEPSEG		TGAGGAAGGTAGCCCGGCTGGT
  	SAPGSPAGSPTSTE		TCTCCGACTTCTACTGAGGAAG
  	EGTSESATPESGPG		GTACTTCTACCGAACCTTCCGA
  	SEPATSGSETPGSS		AGGTAGCGCTCCAGGTCCAGAA
  	TPSGATGSPGASPG		CCAACGGGGCCGGCCCCAAGCG
  	TSSTGSPGSSTPSG		GAGGTAGCGAACCGGCAACCTC
  	ATGSPGSTSESPSG		CGGCTCTGAAACCCCAGGTACC
  	TAPGTSPSGESSTA		TCTGAAAGCGCTACTCCTGAAT
  	PGSTSSTAESPGPG		CCGGCCCAGGTAGCCCGGCAGG
  	SSTPSGATGSPGAS		TTCTCCGACTTCCACTGAGGAA
  	PGTSSTGSPGTPGS		GGTACTTCTGAAAGCGCTACTC
  	GTASSSPGSPAGSP		CTGAGTCCGGCCCAGGTAGCCC
  	TSTEEGSPAGSPTS		GGCTGGCTCTCCGACTTCCACC
  	TEEGTSTEPSEGSAP		GAGGAAGGTAGCCCGGCTGGCT
  			CTCCAACTTCTACTGAAGAAGG
  			TACTTCTGAAAGCGCTACTCCT
  			GAGTCCGGCCCAGGTAGCCCGG
  			CTGGCTCTCCGACTTCCACCGA
  			GGAAGGTAGCCCGGCTGGCTCT
  			CCAACTTCTACTGAAGAAGGTT
  			CTACCAGCTCTACCGCTGAATC
  			TCCTGGCCCAGGTTCTACTAGC
  			GAATCTCCGTCTGGCACCGCAC
  			CAGGTACTTCCCCTAGCGGTGA
  			ATCTTCTACTGCACCAGGTTCTA
  			CCAGCGAATCTCCTTCTGGCAC
  			CGCTCCAGGTTCTACTAGCGAA
  			TCCCCGTCTGGTACCGCACCAG
  			GTACTTCTCCTAGCGGCGAATC
  			TTCTACCGCACCAGGTACTTCT
  			ACCGAACCTTCCGAGGGCAGCG
  			CACCAGGTACTTCTGAAAGCGC
  			TACCCCTGAGTCCGGCCCAGGT
  			ACTTCTGAAAGCGCTACTCCTG
  			AATCCGGTCCAGGTAGCGAACC
  			GGCAACCTCTGGCTCTGAAACC
  			CCAGGTACCTCTGAAAGCGCTA
  			CTCCGGAATCTGGTCCAGGTAC
  			TTCTGAAAGCGCTACTCCGGAA
  			TCCGGTCCAGGTACCTCTACTG
  			AACCTTCTGAGGGCAGCGCTCC
  			AGGTACTTCTGAAAGCGCTACC
  			CCGGAGTCCGGTCCAGGTACTT
  			CTACTGAACCGTCCGAAGGTAG
  			CGCACCAGGTACCTCCCCTAGC
  			GGCGAATCTTCTACTGCTCCAG
  			GTACCTCTCCTAGCGGCGAATC
  			TTCTACCGCTCCAGGTACCTCCC
  			CTAGCGGTGAATCTTCTACCGC
  			ACCAGGTACTTCTACCGAACCG
  			TCCGAGGGTAGCGCACCAGGTA
  			GCCCAGCAGGTTCTCCTACCTC
  			CACCGAGGAAGGTACTTCTACC
  			GAACCGTCCGAGGGTAGCGCAC
  			CAGGTTCTAGCCCTTCTGCTTCC
  			ACCGGTACCGGCCCAGGTAGCT
  			CTACTCCGTCTGGTGCAACTGG
  			CTCTCCAGGTAGCTCTACTCCGT
  			CTGGTGCAACCGGCTCCCCAGG
  			TAGCTCTACCCCGTCTGGTGCT
  			ACCGGCTCTCCAGGTAGCTCTA
  			CCCCGTCTGGTGCAACCGGCTC
  			CCCAGGTGCATCCCCGGGTACT
  			AGCTCTACCGGTTCTCCAGGTG
  			CAAGCGCAAGCGGCGCGCCAA
  			GCACGGGAGGTACTTCTCCGAG
  			CGGTGAATCTTCTACCGCACCA
  			GGTTCTACTAGCTCTACCGCTG
  			AATCTCCGGGCCCAGGTACTTC
  			TCCGAGCGGTGAATCTTCTACT
  			GCTCCAGGTACCTCTGAAAGCG
  			CTACTCCGGAGTCTGGCCCAGG
  			TACCTCTACTGAACCGTCTGAG
  			GGTAGCGCTCCAGGTACTTCTA
  			CTGAACCGTCCGAAGGTAGCGC
  			ACCAGGTTCTAGCCCTTCTGCA
  			TCTACTGGTACTGGCCCAGGTA
  			GCTCTACTCCTTCTGGTGCTACC
  			GGCTCTCCAGGTGCTTCTCCGG
  			GTACTAGCTCTACCGGTTCTCC
  			AGGTACTTCTACTCCGGAAAGC
  			GGTTCCGCATCTCCAGGTACTT
  			CTCCTAGCGGTGAATCTTCTACT
  			GCTCCAGGTACCTCTCCTAGCG
  			GCGAATCTTCTACTGCTCCAGG
  			TACTTCTGAAAGCGCAACCCCT
  			GAATCCGGTCCAGGTAGCGAAC
  			CGGCTACTTCTGGCTCTGAGAC
  			TCCAGGTACTTCTACCGAACCG
  			TCCGAAGGTAGCGCACCAGGTT
  			CTACCAGCGAATCCCCTTCTGG
  			TACTGCTCCAGGTTCTACCAGC
  			GAATCCCCTTCTGGCACCGCAC
  			CAGGTACTTCTACCCCTGAAAG
  			CGGCTCCGCTTCTCCAGGTAGC
  			CCGGCAGGCTCTCCGACCTCTA
  			CTGAGGAAGGTACTTCTGAAAG
  			CGCAACCCCGGAGTCCGGCCCA
  			GGTACCTCTACCGAACCGTCTG
  			AGGGCAGCGCACCAGGTAGCCC
  			TGCTGGCTCTCCAACCTCCACC
  			GAAGAAGGTACCTCTGAAAGCG
  			CAACCCCTGAATCCGGCCCAGG
  			TAGCGAACCGGCAACCTCCGGT
  			TCTGAAACCCCAGGTAGCTCTA
  			CCCCGTCTGGTGCTACCGGTTC
  			CCCAGGTGCTTCTCCTGGTACT
  			AGCTCTACCGGTTCTCCAGGTA
  			GCTCTACCCCGTCTGGTGCTACT
  			GGCTCTCCAGGTTCTACTAGCG
  			AATCCCCGTCTGGTACTGCTCC
  			AGGTACTTCCCCTAGCGGTGAA
  			TCTTCTACTGCTCCAGGTTCTAC
  			CAGCTCTACCGCAGAATCTCCG
  			GGTCCAGGTAGCTCTACCCCTT
  			CTGGTGCAACCGGCTCTCCAGG
  			TGCATCCCCGGGTACCAGCTCT
  			ACCGGTTCTCCAGGTACTCCGG
  			GTAGCGGTACCGCTTCTTCCTCT
  			CCAGGTAGCCCTGCTGGCTCTC
  			CGACTTCTACTGAGGAAGGTAG
  			CCCGGCTGGTTCTCCGACTTCTA
  			CTGAGGAAGGTACTTCTACCGA
  			ACCTTCCGAAGGTAGCGCTCCA
  *Sequence name reflects N- to C-terminus configuration of the coagulation factor and XTEN components

• TABLE 42
  Exemplary CFXTEN comprising CF, cleavage sequences
  and XTEN sequences

  		SEQ		SEQ
  CFXTEN		ID		ID
  Name*	Amino Acid Sequence	NO:	DNA Nucleotide Sequence	NO:
  FVII-	ANAFLEELRPGSLE	702	GCCAACGCGTTCCTGGAGGAGC	703
  FXIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE288	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	KLTRAETGGTSES		AGGTCATCATCCCCAGCACGTA
  	ATPESGPGSEPATS		CGTCCCGGGCACCACCAACCAC
  	GSETPGTSESATPE		GACATCGCGCTGCTCCGCCTGC
  	SGPGSEPATSGSET		ACCAGCCCGTGGTCCTCACTGA
  	PGTSESATPESGPG		CCATGTGGTGCCCCTCTGCCTG
  	TSTEPSEGSAPGSP		CCCGAACGGACGTTCTCTGAGA
  	AGSPTSTEEGTSES		GGACGCTGGCCTTCGTGCGCTT
  	ATPESGPGSEPATS		CTCATTGGTCAGCGGCTGGGGC
  	GSETPGTSESATPE		CAGCTGCTGGACCGTGGCGCCA
  	SGPGSPAGSPTSTE		CGGCCCTGGAGCTCATGGTCCT
  	EGSPAGSPTSTEEG		CAACGTGCCCCGGCTGATGACC
  	TSTEPSEGSAPGTS		CAGGACTGCCTGCAGCAGTCAC
  	ESATPESGPGTSES		GGAAGGTGGGAGACTCCCCAA
  	ATPESGPGTSESAT		ATATCACGGAGTACATGTTCTG
  	PESGPGSEPATSGS		TGCCGGCTACTCGGATGGCAGC
  	ETPGSEPATSGSET		AAGGACTCCTGCAAGGGGGAC
  	PGSPAGSPTSTEEG		AGTGGAGGCCCACATGCCACCC
  	TSTEPSEGSAPGTS		ACTACCGGGGCACGTGGTACCT
  	TEPSEGSAPGSEPA		GACGGGCATCGTCAGCTGGGGC
  	TSGSETPGTSESAT		CAGGGCTGCGCAACCGTGGGCC
  	PESGPGTSTEPSEG		ACTTTGGGGTGTACACCAGGGT
  	SAP		CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-	ANAFLEELRPGSLE	704	GCCAACGCGTTCCTGGAGGAGC	705
  FXIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE864	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	KLTRAETGGSPAG		AGGTCATCATCCCCAGCACGTA
  	SPTSTEEGTSESAT		CGTCCCGGGCACCACCAACCAC
  	PESGPGTSTEPSEG		GACATCGCGCTGCTCCGCCTGC
  	SAPGSPAGSPTSTE		ACCAGCCCGTGGTCCTCACTGA
  	EGTSTEPSEGSAPG		CCATGTGGTGCCCCTCTGCCTG
  	TSTEPSEGSAPGTS		CCCGAACGGACGTTCTCTGAGA
  	ESATPESGPGSEPA		GGACGCTGGCCTTCGTGCGCTT
  	TSGSETPGSEPATS		CTCATTGGTCAGCGGCTGGGGC
  	GSETPGSPAGSPTS		CAGCTGCTGGACCGTGGCGCCA
  	TEEGTSESATPESG		CGGCCCTGGAGCTCATGGTCCT
  	PGTSTEPSEGSAPG		CAACGTGCCCCGGCTGATGACC
  	TSTEPSEGSAPGSP		CAGGACTGCCTGCAGCAGTCAC
  	AGSPTSTEEGTSTE		GGAAGGTGGGAGACTCCCCAA
  	PSEGSAPGTSTEPS		ATATCACGGAGTACATGTTCTG
  	EGSAPGTSESATPE		TGCCGGCTACTCGGATGGCAGC
  	SGPGTSTEPSEGSA		AAGGACTCCTGCAAGGGGGAC
  	PGTSESATPESGPG		AGTGGAGGCCCACATGCCACCC
  	SEPATSGSETPGTS		ACTACCGGGGCACGTGGTACCT
  	TEPSEGSAPGTSTE		GACGGGCATCGTCAGCTGGGGC
  	PSEGSAPGTSESAT		CAGGGCTGCGCAACCGTGGGCC
  	PESGPGTSESATPE		ACTTTGGGGTGTACACCAGGGT
  	SGPGSPAGSPTSTE		CTCCCAGTACATCGAGTGGCTG
  	EGTSESATPESGPG		CAAAAGCTCATGCGCTCAGAGC
  	SEPATSGSETPGTS		CACGCCCAGGAGTCCTCCTGCG
  	ESATPESGPGTSTE		AGCCCCATTTCCCGGT0GGTGGT
  	PSEGSAPGTSTEPS		AGCCCGGCTGGCTCTCCTACCT
  	EGSAPGTSTEPSEG		CTACTGAGGAAGGTACTTCTGA
  	SAPGTSTEPSEGSA		AAGCGCTACTCCTGAGTCTGGT
  	PGTSTEPSEGSAPG		CCAGGTACCTCTACTGAACCGT
  	TSTEPSEGSAPGSP		CCGAAGGTAGCGCTCCAGGTAG
  	AGSPTSTEEGTSTE		CCCAGCAGGCTCTCCGACTTCC
  	PSEGSAPGTSESAT		ACTGAGGAAGGTACTTCTACTG
  	PESGPGSEPATSGS		AACCTTCCGAAGGCAGCGCACC
  	ETPGTSESATPESG		AGGTACCTCTACTGAACCTTCT
  	PGSEPATSGSETPG		GAGGGCAGCGCTCCAGGTACTT
  	TSESATPESGPGTS		CTGAAAGCGCTACCCCGGAATC
  	TEPSEGSAPGTSES		TGGCCCAGGTAGCGAACCGGCT
  	ATPESGPGSPAGSP		ACTTCTGGTTCTGAAACCCCAG
  	TSTEEGSPAGSPTS		GTAGCGAACCGGCTACCTCCGG
  	TEEGSPAGSPTSTE		TTCTGAAACTCCAGGTAGCCCG
  	EGTSESATPESGPG		GCAGGCTCTCCGACCTCTACTG
  	TSTEPSEGSAPGTS		AGGAAGGTACTTCTGAAAGCGC
  	ESATPESGPGSEPA		AACCCCGGAGTCCGGCCCAGGT
  	TSGSETPGTSESAT		ACCTCTACCGAACCGTCTGAGG
  	PESGPGSEPATSGS		GCAGCGCACCAGGTACTTCTAC
  	ETPGTSESATPESG		CGAACCGTCCGAGGGTAGCGCA
  	PGTSTEPSEGSAPG		CCAGGTAGCCCAGCAGGTTCTC
  	SPAGSPTSTEEGTS		CTACCTCCACCGAGGAAGGTAC
  	ESATPESGPGSEPA		TTCTACCGAACCGTCCGAGGGT
  	TSGSETPGTSESAT		AGCGCACCAGGTACCTCTACTG
  	PESGPGSPAGSPTS		AACCTTCTGAGGGCAGCGCTCC
  	TEEGSPAGSPTSTE		AGGTACTTCTGAAAGCGCTACC
  	EGTSTEPSEGSAPG		CCGGAGTCCGGTCCAGGTACTT
  	TSESATPESGPGTS		CTACTGAACCGTCCGAAGGTAG
  	ESATPESGPGTSES		CGCACCAGGTACTTCTGAAAGC
  	ATPESGPGSEPATS		GCAACCCCTGAATCCGGTCCAG
  	GSETPGSEPATSGS		GTAGCGAACCGGCTACTTCTGG
  	ETPGSPAGSPTSTE		CTCTGAGACTCCAGGTACTTCT
  	EGTSTEPSEGSAPG		ACCGAACCGTCCGAAGGTAGCG
  	TSTEPSEGSAPGSE		CACCAGGTACTTCTACTGAACC
  	PATSGSETPGTSES		GTCTGAAGGTAGCGCACCAGGT
  	ATPESGPGTSTEPS		ACTTCTGAAAGCGCAACCCCGG
  	EGSAP		AATCCGGCCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAGTCCGGC
  			CCAGGTAGCCCTGCTGGCTCTC
  			CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	706	TATAATTCAGGTAAATTGGAAG	707
  FXIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE288	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGK		AATTACAGTTGTCGCAGGTGAA
  	LTRAETGGTSESA		CATAATATTGAGGAGACAGAAC
  	TPESGPGSEPATSG		ATACAGAGCAAAAGCGAAATG
  	SETPGTSESATPES		TGATTCGAATTATTCCTCACCAC
  	GPGSEPATSGSETP		AACTACAATGCAGCTATTAATA
  	GTSESATPESGPGT		AGTACAACCATGACATTGCCCT
  	STEPSEGSAPGSPA		TCTGGAACTGGACGAACCCTTA
  	GSPTSTEEGTSESA		GTGCTAAACAGCTACGTTACAC
  	TPESGPGSEPATSG		CTATTTGCATTGCTGACAAGGA
  	SETPGTSESATPES		ATACACGAACATCTTCCTCAAA
  	GPGSPAGSPTSTEE		TTTGGATCTGGCTATGTAAGTG
  	GSPAGSPTSTEEGT		GCTGGGGAAGAGTCTTCCACAA
  	STEPSEGSAPGTSE		AGGGAGATCAGCTTTAGTTCTT
  	SATPESGPGTSESA		CAGTACCTTAGAGTTCCACTTG
  	TPESGPGTSESATP		TTGACCGAGCCACATGTCTTCG
  	ESGPGSEPATSGSE		ATCTACAAAGTTCACCATCTAT
  	TPGSEPATSGSETP		AACAACATGTTCTGTGCTGGCT
  	GSPAGSPTSTEEGT		TCCATGAAGGAGGTAGAGATTC
  	STEPSEGSAPGTST		ATGTCAAGGAGATAGTGGGGG
  	EPSEGSAPGSEPAT		ACCCCATGTTACTGAAGTGGAA
  	SGSETPGTSESATP		GGGACCAGTTTCTTAACTGGAA
  	ESGPGTSTEPSEGS		TTATTAGCTGGGGTGAAGAGTG
  	AP		TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0GGT
  			GGTACCTCTGAAAGCGCAACTC
  			CTGAGTCTGGCCCAGGTAGCGA
  			ACCTGCTACCTCCGGCTCTGAG
  			ACTCCAGGTACCTCTGAAAGCG
  			CAACCCCGGAATCTGGTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCAGGTA
  			GCCCTGCTGGCTCTCCAACCTC
  			CACCGAAGAAGGTACCTCTGAA
  			AGCGCAACCCCTGAATCCGGCC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACCCCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTACTTC
  			TACCGAACCTTCCGAGGGCAGC
  			GCACCAGGTACTTCTGAAAGCG
  			CTACCCCTGAGTCCGGCCCAGG
  			TACTTCTGAAAGCGCTACTCCT
  			GAATCCGGTCCAGGTACTTCTG
  			AAAGCGCTACCCCGGAATCTGG
  			CCCAGGTAGCGAACCGGCTACT
  			TCTGGTTCTGAAACCCCAGGTA
  			GCGAACCGGCTACCTCCGGTTC
  			TGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGG
  			AAGGTACTTCTACTGAACCTTC
  			CGAAGGCAGCGCACCAGGTACC
  			TCTACTGAACCTTCTGAGGGCA
  			GCGCTCCAGGTAGCGAACCTGC
  			AACCTCTGGCTCTGAAACCCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CTGAATCTGGCCCAGGTACTTC
  			TACTGAACCGTCCGAGGGCAGC
  			GCACCA
  FIX-	YNSGKLEEFVQGN	708	TATAATTCAGGTAAATTGGAAG	709
  FXIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE864	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGK		AATTACAGTTGTCGCAGGTGAA
  	LTRAETGGSPAGS		CATAATATTGAGGAGACAGAAC
  	PTSTEEGTSESATP		ATACAGAGCAAAAGCGAAATG
  	ESGPGTSTEPSEGS		TGATTCGAATTATTCCTCACCAC
  	APGSPAGSPTSTEE		AACTACAATGCAGCTATTAATA
  	GTSTEPSEGSAPGT		AGTACAACCATGACATTGCCCT
  	STEPSEGSAPGTSE		TCTGGAACTGGACGAACCCTTA
  	SATPESGPGSEPAT		GTGCTAAACAGCTACGTTACAC
  	SGSETPGSEPATSG		CTATTTGCATTGCTGACAAGGA
  	SETPGSPAGSPTST		ATACACGAACATCTTCCTCAAA
  	EEGTSESATPESGP		TTTGGATCTGGCTATGTAAGTG
  	GTSTEPSEGSAPGT		GCTGGGGAAGAGTCTTCCACAA
  	STEPSEGSAPGSPA		AGGGAGATCAGCTTTAGTTCTT
  	GSPTSTEEGTSTEP		CAGTACCTTAGAGTTCCACTTG
  	SEGSAPGTSTEPSE		TTGACCGAGCCACATGTCTTCG
  	GSAPGTSESATPES		ATCTACAAAGTTCACCATCTAT
  	GPGTSTEPSEGSAP		AACAACATGTTCTGTGCTGGCT
  	GTSESATPESGPGS		TCCATGAAGGAGGTAGAGATTC
  	EPATSGSETPGTST		ATGTCAAGGAGATAGTGGGGG
  	EPSEGSAPGTSTEP		ACCCCATGTTACTGAAGTGGAA
  	SEGSAPGTSESATP		GGGACCAGTTTCTTAACTGGAA
  	ESGPGTSESATPES		TTATTAGCTGGGGTGAAGAGTG
  	GPGSPAGSPTSTEE		TGCAATGAAAGGCAAATATGGA
  	GTSESATPESGPGS		ATATATACCAAGGTATCCCGGT
  	EPATSGSETPGTSE		ATGTCAACTGGATTAAGGAAAA
  	SATPESGPGTSTEP		AACAAAGCTCACTGGGGT0GGT
  	SEGSAPGTSTEPSE		GGTAGCCCGGCTGGCTCTCCTA
  	GSAPGTSTEPSEGS		CCTCTACTGAGGAAGGTACTTC
  	APGTSTEPSEGSAP		TGAAAGCGCTACTCCTGAGTCT
  	GTSTEPSEGSAPGT		GGTCCAGGTACCTCTACTGAAC
  	STEPSEGSAPGSPA		CGTCCGAAGGTAGCGCTCCAGG
  	GSPTSTEEGTSTEP		TAGCCCAGCAGGCTCTCCGACT
  	SEGSAPGTSESATP		TCCACTGAGGAAGGTACTTCTA
  	ESGPGSEPATSGSE		CTGAACCTTCCGAAGGCAGCGC
  	TPGTSESATPESGP		ACCAGGTACCTCTACTGAACCT
  	GSEPATSGSETPGT		TCTGAGGGCAGCGCTCCAGGTA
  	SESATPESGPGTST		CTTCTGAAAGCGCTACCCCGGA
  	EPSEGSAPGTSESA		ATCTGGCCCAGGTAGCGAACCG
  	TPESGPGSPAGSPT		GCTACTTCTGGTTCTGAAACCC
  	STEEGSPAGSPTST		CAGGTAGCGAACCGGCTACCTC
  	EEGSPAGSPTSTEE		CGGTTCTGAAACTCCAGGTAGC
  	GTSESATPESGPGT		CCGGCAGGCTCTCCGACCTCTA
  	STEPSEGSAPGTSE		CTGAGGAAGGTACTTCTGAAAG
  	SATPESGPGSEPAT		CGCAACCCCGGAGTCCGGCCCA
  	SGSETPGTSESATP		GGTACCTCTACCGAACCGTCTG
  	ESGPGSEPATSGSE		AGGGCAGCGCACCAGGTACTTC
  	TPGTSESATPESGP		TACCGAACCGTCCGAGGGTAGC
  	GTSTEPSEGSAPGS		GCACCAGGTAGCCCAGCAGGTT
  	PAGSPTSTEEGTSE		CTCCTACCTCCACCGAGGAAGG
  	SATPESGPGSEPAT		TACTTCTACCGAACCGTCCGAG
  	SGSETPGTSESATP		GGTAGCGCACCAGGTACCTCTA
  	ESGPGSPAGSPTST		CTGAACCTTCTGAGGGCAGCGC
  	EEGSPAGSPTSTEE		TCCAGGTACTTCTGAAAGCGCT
  	GTSTEPSEGSAPGT		ACCCCGGAGTCCGGTCCAGGTA
  	SESATPESGPGTSE		CTTCTACTGAACCGTCCGAAGG
  	SATPESGPGTSESA		TAGCGCACCAGGTACTTCTGAA
  	TPESGPGSEPATSG		AGCGCAACCCCTGAATCCGGTC
  	SETPGSEPATSGSE		CAGGTAGCGAACCGGCTACTTC
  	TPGSPAGSPTSTEE		TGGCTCTGAGACTCCAGGTACT
  	GTSTEPSEGSAPGT		TCTACCGAACCGTCCGAAGGTA
  	STEPSEGSAPGSEP		GCGCACCAGGTACTTCTACTGA
  	ATSGSETPGTSESA		ACCGTCTGAAGGTAGCGCACCA
  	TPESGPGTSTEPSE		GGTACTTCTGAAAGCGCAACCC
  	GSAP		CGGAATCCGGCCCAGGTACCTC
  			TGAAAGCGCAACCCCGGAGTCC
  			GGCCCAGGTAGCCCTGCTGGCT
  			CTCCAACCTCCACCGAAGAAGG
  			TACCTCTGAAAGCGCAACCCCT
  			GAATCCGGCCCAGGTAGCGAAC
  			CGGCAACCTCCGGTTCTGAAAC
  			CCCAGGTACCTCTGAAAGCGCT
  			ACTCCGGAGTCTGGCCCAGGTA
  			CCTCTACTGAACCGTCTGAGGG
  			TAGCGCTCCAGGTACTTCTACT
  			GAACCGTCCGAAGGTAGCGCAC
  			CAGGTACTTCTACCGAACCGTC
  			CGAAGGCAGCGCTCCAGGTACC
  			TCTACTGAACCTTCCGAGGGCA
  			GCGCTCCAGGTACCTCTACCGA
  			ACCTTCTGAAGGTAGCGCACCA
  			GGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTAGCCC
  			AGCAGGTTCTCCTACCTCCACC
  			GAGGAAGGTACTTCTACCGAAC
  			CGTCCGAGGGTAGCGCACCAGG
  			TACCTCTGAAAGCGCAACTCCT
  			GAGTCTGGCCCAGGTAGCGAAC
  			CTGCTACCTCCGGCTCTGAGAC
  			TCCAGGTACCTCTGAAAGCGCA
  			ACCCCGGAATCTGGTCCAGGTA
  			GCGAACCTGCAACCTCTGGCTC
  			TGAAACCCCAGGTACCTCTGAA
  			AGCGCTACTCCTGAATCTGGCC
  			CAGGTACTTCTACTGAACCGTC
  			CGAGGGCAGCGCACCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTAGCCC
  			GGCAGGCTCTCCGACCTCTACT
  			GAGGAAGGTACTTCTGAAAGCG
  			CAACCCCGGAGTCCGGCCCAGG
  			TACCTCTACCGAACCGTCTGAG
  			GGCAGCGCACCAGGTACCTCTG
  			AAAGCGCAACTCCTGAGTCTGG
  			CCCAGGTAGCGAACCTGCTACC
  			TCCGGCTCTGAGACTCCAGGTA
  			CCTCTGAAAGCGCAACCCCGGA
  			ATCTGGTCCAGGTAGCGAACCT
  			GCAACCTCTGGCTCTGAAACCC
  			CAGGTACCTCTGAAAGCGCTAC
  			TCCTGAATCTGGCCCAGGTACT
  			TCTACTGAACCGTCCGAGGGCA
  			GCGCACCAGGTAGCCCTGCTGG
  			CTCTCCAACCTCCACCGAAGAA
  			GGTACCTCTGAAAGCGCAACCC
  			CTGAATCCGGCCCAGGTAGCGA
  			ACCGGCAACCTCCGGTTCTGAA
  			ACCCCAGGTACTTCTGAAAGCG
  			CTACTCCTGAGTCCGGCCCAGG
  			TAGCCCGGCTGGCTCTCCGACT
  			TCCACCGAGGAAGGTAGCCCGG
  			CTGGCTCTCCAACTTCTACTGA
  			AGAAGGTACTTCTACCGAACCT
  			TCCGAGGGCAGCGCACCAGGTA
  			CTTCTGAAAGCGCTACCCCTGA
  			GTCCGGCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAATCCGGTC
  			CAGGTACTTCTGAAAGCGCTAC
  			CCCGGAATCTGGCCCAGGTAGC
  			GAACCGGCTACTTCTGGTTCTG
  			AAACCCCAGGTAGCGAACCGGC
  			TACCTCCGGTTCTGAAACTCCA
  			GGTAGCCCAGCAGGCTCTCCGA
  			CTTCCACTGAGGAAGGTACTTC
  			TACTGAACCTTCCGAAGGCAGC
  			GCACCAGGTACCTCTACTGAAC
  			CTTCTGAGGGCAGCGCTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	710	GCCAACGCGTTCCTGGAGGAGC	711
  FXIIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE288	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	TMTRIVGGGGTSE		AGGTCATCATCCCCAGCACGTA
  	SATPESGPGSEPAT		CGTCCCGGGCACCACCAACCAC
  	SGSETPGTSESATP		GACATCGCGCTGCTCCGCCTGC
  	ESGPGSEPATSGSE		ACCAGCCCGTGGTCCTCACTGA
  	TPGTSESATPESGP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGS		CCCGAACGGACGTTCTCTGAGA
  	PAGSPTSTEEGTSE		GGACGCTGGCCTTCGTGCGCTT
  	SATPESGPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGTSESATP		CAGCTGCTGGACCGTGGCGCCA
  	ESGPGSPAGSPTST		CGGCCCTGGAGCTCATGGTCCT
  	EEGSPAGSPTSTEE		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGT		CAGGACTGCCTGCAGCAGTCAC
  	SESATPESGPGTSE		GGAAGGTGGGAGACTCCCCAA
  	SATPESGPGTSESA		ATATCACGGAGTACATGTTCTG
  	TPESGPGSEPATSG		TGCCGGCTACTCGGATGGCAGC
  	SETPGSEPATSGSE		AAGGACTCCTGCAAGGGGGAC
  	TPGSPAGSPTSTEE		AGTGGAGGCCCACATGCCACCC
  	GTSTEPSEGSAPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGSEP		GACGGGCATCGTCAGCTGGGGC
  	ATSGSETPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSTEPSE		ACTTTGGGGTGTACACCAGGGT
  	GSAP		CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-	ANAFLEELRPGSLE	712	GCCAACGCGTTCCTGGAGGAGC	713
  FXIIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE864	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	TMTRIVGGGGSPA		AGGTCATCATCCCCAGCACGTA
  	GSPTSTEEGTSESA		CGTCCCGGGCACCACCAACCAC
  	TPESGPGTSTEPSE		GACATCGCGCTGCTCCGCCTGC
  	GSAPGSPAGSPTST		ACCAGCCCGTGGTCCTCACTGA
  	EEGTSTEPSEGSAP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGT		CCCGAACGGACGTTCTCTGAGA
  	SESATPESGPGSEP		GGACGCTGGCCTTCGTGCGCTT
  	ATSGSETPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGSPAGSPT		CAGCTGCTGGACCGTGGCGCCA
  	STEEGTSESATPES		CGGCCCTGGAGCTCATGGTCCT
  	GPGTSTEPSEGSAP		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGS		CAGGACTGCCTGCAGCAGTCAC
  	PAGSPTSTEEGTST		GGAAGGTGGGAGACTCCCCAA
  	EPSEGSAPGTSTEP		ATATCACGGAGTACATGTTCTG
  	SEGSAPGTSESATP		TGCCGGCTACTCGGATGGCAGC
  	ESGPGTSTEPSEGS		AAGGACTCCTGCAAGGGGGAC
  	APGTSESATPESGP		AGTGGAGGCCCACATGCCACCC
  	GSEPATSGSETPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGTST		GACGGGCATCGTCAGCTGGGGC
  	EPSEGSAPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSESATP		ACTTTGGGGTGTACACCAGGGT
  	ESGPGSPAGSPTST		CTCCCAGTACATCGAGTGGCTG
  	EEGTSESATPESGP		CAAAAGCTCATGCGCTCAGAGC
  	GSEPATSGSETPGT		CACGCCCAGGAGTCCTCCTGCG
  	SESATPESGPGTST		AGCCCCATTTCCCGGT0GGTGGT
  	EPSEGSAPGTSTEP		AGCCCGGCTGGCTCTCCTACCT
  	SEGSAPGTSTEPSE		CTACTGAGGAAGGTACTTCTGA
  	GSAPGTSTEPSEGS		AAGCGCTACTCCTGAGTCTGGT
  	APGTSTEPSEGSAP		CCAGGTACCTCTACTGAACCGT
  	GTSTEPSEGSAPGS		CCGAAGGTAGCGCTCCAGGTAG
  	PAGSPTSTEEGTST		CCCAGCAGGCTCTCCGACTTCC
  	EPSEGSAPGTSESA		ACTGAGGAAGGTACTTCTACTG
  	TPESGPGSEPATSG		AACCTTCCGAAGGCAGCGCACC
  	SETPGTSESATPES		AGGTACCTCTACTGAACCTTCT
  	GPGSEPATSGSETP		GAGGGCAGCGCTCCAGGTACTT
  	GTSESATPESGPGT		CTGAAAGCGCTACCCCGGAATC
  	STEPSEGSAPGTSE		TGGCCCAGGTAGCGAACCGGCT
  	SATPESGPGSPAGS		ACTTCTGGTTCTGAAACCCCAG
  	PTSTEEGSPAGSPT		GTAGCGAACCGGCTACCTCCGG
  	STEEGSPAGSPTST		TTCTGAAACTCCAGGTAGCCCG
  	EEGTSESATPESGP		GCAGGCTCTCCGACCTCTACTG
  	GTSTEPSEGSAPGT		AGGAAGGTACTTCTGAAAGCGC
  	SESATPESGPGSEP		AACCCCGGAGTCCGGCCCAGGT
  	ATSGSETPGTSESA		ACCTCTACCGAACCGTCTGAGG
  	TPESGPGSEPATSG		GCAGCGCACCAGGTACTTCTAC
  	SETPGTSESATPES		CGAACCGTCCGAGGGTAGCGCA
  	GPGTSTEPSEGSAP		CCAGGTAGCCCAGCAGGTTCTC
  	GSPAGSPTSTEEGT		CTACCTCCACCGAGGAAGGTAC
  	SESATPESGPGSEP		TTCTACCGAACCGTCCGAGGGT
  	ATSGSETPGTSESA		AGCGCACCAGGTACCTCTACTG
  	TPESGPGSPAGSPT		AACCTTCTGAGGGCAGCGCTCC
  	STEEGSPAGSPTST		AGGTACTTCTGAAAGCGCTACC
  	EEGTSTEPSEGSAP		CCGGAGTCCGGTCCAGGTACTT
  	GTSESATPESGPGT		CTACTGAACCGTCCGAAGGTAG
  	SESATPESGPGTSE		CGCACCAGGTACTTCTGAAAGC
  	SATPESGPGSEPAT		GCAACCCCTGAATCCGGTCCAG
  	SGSETPGSEPATSG		GTAGCGAACCGGCTACTTCTGG
  	SETPGSPAGSPTST		CTCTGAGACTCCAGGTACTTCT
  	EEGTSTEPSEGSAP		ACCGAACCGTCCGAAGGTAGCG
  	GTSTEPSEGSAPGS		CACCAGGTACTTCTACTGAACC
  	EPATSGSETPGTSE		GTCTGAAGGTAGCGCACCAGGT
  	SATPESGPGTSTEP		ACTTCTGAAAGCGCAACCCCGG
  	SEGSAP		AATCCGGCCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAGTCCGGC
  			CCAGGTAGCCCTGCTGGCTCTC
  			CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	714	TATAATTCAGGTAAATTGGAAG	715
  FXIIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE288	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGT		AATTACAGTTGTCGCAGGTGAA
  	MTRIVGGGGTSES		CATAATATTGAGGAGACAGAAC
  	ATPESGPGSEPATS		ATACAGAGCAAAAGCGAAATG
  	GSETPGTSESATPE		TGATTCGAATTATTCCTCACCAC
  	SGPGSEPATSGSET		AACTACAATGCAGCTATTAATA
  	PGTSESATPESGPG		AGTACAACCATGACATTGCCCT
  	TSTEPSEGSAPGSP		TCTGGAACTGGACGAACCCTTA
  	AGSPTSTEEGTSES		GTGCTAAACAGCTACGTTACAC
  	ATPESGPGSEPATS		CTATTTGCATTGCTGACAAGGA
  	GSETPGTSESATPE		ATACACGAACATCTTCCTCAAA
  	SGPGSPAGSPTSTE		TTTGGATCTGGCTATGTAAGTG
  	EGSPAGSPTSTEEG		GCTGGGGAAGAGTCTTCCACAA
  	TSTEPSEGSAPGTS		AGGGAGATCAGCTTTAGTTCTT
  	ESATPESGPGTSES		CAGTACCTTAGAGTTCCACTTG
  	ATPESGPGTSESAT		TTGACCGAGCCACATGTCTTCG
  	PESGPGSEPATSGS		ATCTACAAAGTTCACCATCTAT
  	ETPGSEPATSGSET		AACAACATGTTCTGTGCTGGCT
  	PGSPAGSPTSTEEG		TCCATGAAGGAGGTAGAGATTC
  	TSTEPSEGSAPGTS		ATGTCAAGGAGATAGTGGGGG
  	TEPSEGSAPGSEPA		ACCCCATGTTACTGAAGTGGAA
  	TSGSETPGTSESAT		GGGACCAGTTTCTTAACTGGAA
  	PESGPGTSTEPSEG		TTATTAGCTGGGGTGAAGAGTG
  	SAP		TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0GGT
  			GGTACCTCTGAAAGCGCAACTC
  			CTGAGTCTGGCCCAGGTAGCGA
  			ACCTGCTACCTCCGGCTCTGAG
  			ACTCCAGGTACCTCTGAAAGCG
  			CAACCCCGGAATCTGGTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCAGGTA
  			GCCCTGCTGGCTCTCCAACCTC
  			CACCGAAGAAGGTACCTCTGAA
  			AGCGCAACCCCTGAATCCGGCC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACCCCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTACTTC
  			TACCGAACCTTCCGAGGGCAGC
  			GCACCAGGTACTTCTGAAAGCG
  			CTACCCCTGAGTCCGGCCCAGG
  			TACTTCTGAAAGCGCTACTCCT
  			GAATCCGGTCCAGGTACTTCTG
  			AAAGCGCTACCCCGGAATCTGG
  			CCCAGGTAGCGAACCGGCTACT
  			TCTGGTTCTGAAACCCCAGGTA
  			GCGAACCGGCTACCTCCGGTTC
  			TGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGG
  			AAGGTACTTCTACTGAACCTTC
  			CGAAGGCAGCGCACCAGGTACC
  			TCTACTGAACCTTCTGAGGGCA
  			GCGCTCCAGGTAGCGAACCTGC
  			AACCTCTGGCTCTGAAACCCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CTGAATCTGGCCCAGGTACTTC
  			TACTGAACCGTCCGAGGGCAGC
  			GCACCA
  FIX-	YNSGKLEEFVQGN	716	TATAATTCAGGTAAATTGGAAG	717
  FXIIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE864	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGT		AATTACAGTTGTCGCAGGTGAA
  	MTRIVGGGGSPAG		CATAATATTGAGGAGACAGAAC
  	SPTSTEEGTSESAT		ATACAGAGCAAAAGCGAAATG
  	PESGPGTSTEPSEG		TGATTCGAATTATTCCTCACCAC
  	SAPGSPAGSPTSTE		AACTACAATGCAGCTATTAATA
  	EGTSTEPSEGSAPG		AGTACAACCATGACATTGCCCT
  	TSTEPSEGSAPGTS		TCTGGAACTGGACGAACCCTTA
  	ESATPESGPGSEPA		GTGCTAAACAGCTACGTTACAC
  	TSGSETPGSEPATS		CTATTTGCATTGCTGACAAGGA
  	GSETPGSPAGSPTS		ATACACGAACATCTTCCTCAAA
  	TEEGTSESATPESG		TTTGGATCTGGCTATGTAAGTG
  	PGTSTEPSEGSAPG		GCTGGGGAAGAGTCTTCCACAA
  	TSTEPSEGSAPGSP		AGGGAGATCAGCTTTAGTTCTT
  	AGSPTSTEEGTSTE		CAGTACCTTAGAGTTCCACTTG
  	PSEGSAPGTSTEPS		TTGACCGAGCCACATGTCTTCG
  	EGSAPGTSESATPE		ATCTACAAAGTTCACCATCTAT
  	SGPGTSTEPSEGSA		AACAACATGTTCTGTGCTGGCT
  	PGTSESATPESGPG		TCCATGAAGGAGGTAGAGATTC
  	SEPATSGSETPGTS		ATGTCAAGGAGATAGTGGGGG
  	TEPSEGSAPGTSTE		ACCCCATGTTACTGAAGTGGAA
  	PSEGSAPGTSESAT		GGGACCAGTTTCTTAACTGGAA
  	PESGPGTSESATPE		TTATTAGCTGGGGTGAAGAGTG
  	SGPGSPAGSPTSTE		TGCAATGAAAGGCAAATATGGA
  	EGTSESATPESGPG		ATATATACCAAGGTATCCCGGT
  	SEPATSGSETPGTS		ATGTCAACTGGATTAAGGAAAA
  	ESATPESGPGTSTE		AACAAAGCTCACTGGGGT0GGT
  	PSEGSAPGTSTEPS		GGTAGCCCGGCTGGCTCTCCTA
  	EGSAPGTSTEPSEG		CCTCTACTGAGGAAGGTACTTC
  	SAPGTSTEPSEGSA		TGAAAGCGCTACTCCTGAGTCT
  	PGTSTEPSEGSAPG		GGTCCAGGTACCTCTACTGAAC
  	TSTEPSEGSAPGSP		CGTCCGAAGGTAGCGCTCCAGG
  	AGSPTSTEEGTSTE		TAGCCCAGCAGGCTCTCCGACT
  	PSEGSAPGTSESAT		TCCACTGAGGAAGGTACTTCTA
  	PESGPGSEPATSGS		CTGAACCTTCCGAAGGCAGCGC
  	ETPGTSESATPESG		ACCAGGTACCTCTACTGAACCT
  	PGSEPATSGSETPG		TCTGAGGGCAGCGCTCCAGGTA
  	TSESATPESGPGTS		CTTCTGAAAGCGCTACCCCGGA
  	TEPSEGSAPGTSES		ATCTGGCCCAGGTAGCGAACCG
  	ATPESGPGSPAGSP		GCTACTTCTGGTTCTGAAACCC
  	TSTEEGSPAGSPTS		CAGGTAGCGAACCGGCTACCTC
  	TEEGSPAGSPTSTE		CGGTTCTGAAACTCCAGGTAGC
  	EGTSESATPESGPG		CCGGCAGGCTCTCCGACCTCTA
  	TSTEPSEGSAPGTS		CTGAGGAAGGTACTTCTGAAAG
  	ESATPESGPGSEPA		CGCAACCCCGGAGTCCGGCCCA
  	TSGSETPGTSESAT		GGTACCTCTACCGAACCGTCTG
  	PESGPGSEPATSGS		AGGGCAGCGCACCAGGTACTTC
  	ETPGTSESATPESG		TACCGAACCGTCCGAGGGTAGC
  	PGTSTEPSEGSAPG		GCACCAGGTAGCCCAGCAGGTT
  	SPAGSPTSTEEGTS		CTCCTACCTCCACCGAGGAAGG
  	ESATPESGPGSEPA		TACTTCTACCGAACCGTCCGAG
  	TSGSETPGTSESAT		GGTAGCGCACCAGGTACCTCTA
  	PESGPGSPAGSPTS		CTGAACCTTCTGAGGGCAGCGC
  	TEEGSPAGSPTSTE		TCCAGGTACTTCTGAAAGCGCT
  	EGTSTEPSEGSAPG		ACCCCGGAGTCCGGTCCAGGTA
  	TSESATPESGPGTS		CTTCTACTGAACCGTCCGAAGG
  	ESATPESGPGTSES		TAGCGCACCAGGTACTTCTGAA
  	ATPESGPGSEPATS		AGCGCAACCCCTGAATCCGGTC
  	GSETPGSEPATSGS		CAGGTAGCGAACCGGCTACTTC
  	ETPGSPAGSPTSTE		TGGCTCTGAGACTCCAGGTACT
  	EGTSTEPSEGSAPG		TCTACCGAACCGTCCGAAGGTA
  	TSTEPSEGSAPGSE		GCGCACCAGGTACTTCTACTGA
  	PATSGSETPGTSES		ACCGTCTGAAGGTAGCGCACCA
  	ATPESGPGTSTEPS		GGTACTTCTGAAAGCGCAACCC
  	EGSAP		CGGAATCCGGCCCAGGTACCTC
  			TGAAAGCGCAACCCCGGAGTCC
  			GGCCCAGGTAGCCCTGCTGGCT
  			CTCCAACCTCCACCGAAGAAGG
  			TACCTCTGAAAGCGCAACCCCT
  			GAATCCGGCCCAGGTAGCGAAC
  			CGGCAACCTCCGGTTCTGAAAC
  			CCCAGGTACCTCTGAAAGCGCT
  			ACTCCGGAGTCTGGCCCAGGTA
  			CCTCTACTGAACCGTCTGAGGG
  			TAGCGCTCCAGGTACTTCTACT
  			GAACCGTCCGAAGGTAGCGCAC
  			CAGGTACTTCTACCGAACCGTC
  			CGAAGGCAGCGCTCCAGGTACC
  			TCTACTGAACCTTCCGAGGGCA
  			GCGCTCCAGGTACCTCTACCGA
  			ACCTTCTGAAGGTAGCGCACCA
  			GGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTAGCCC
  			AGCAGGTTCTCCTACCTCCACC
  			GAGGAAGGTACTTCTACCGAAC
  			CGTCCGAGGGTAGCGCACCAGG
  			TACCTCTGAAAGCGCAACTCCT
  			GAGTCTGGCCCAGGTAGCGAAC
  			CTGCTACCTCCGGCTCTGAGAC
  			TCCAGGTACCTCTGAAAGCGCA
  			ACCCCGGAATCTGGTCCAGGTA
  			GCGAACCTGCAACCTCTGGCTC
  			TGAAACCCCAGGTACCTCTGAA
  			AGCGCTACTCCTGAATCTGGCC
  			CAGGTACTTCTACTGAACCGTC
  			CGAGGGCAGCGCACCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTAGCCC
  			GGCAGGCTCTCCGACCTCTACT
  			GAGGAAGGTACTTCTGAAAGCG
  			CAACCCCGGAGTCCGGCCCAGG
  			TACCTCTACCGAACCGTCTGAG
  			GGCAGCGCACCAGGTACCTCTG
  			AAAGCGCAACTCCTGAGTCTGG
  			CCCAGGTAGCGAACCTGCTACC
  			TCCGGCTCTGAGACTCCAGGTA
  			CCTCTGAAAGCGCAACCCCGGA
  			ATCTGGTCCAGGTAGCGAACCT
  			GCAACCTCTGGCTCTGAAACCC
  			CAGGTACCTCTGAAAGCGCTAC
  			TCCTGAATCTGGCCCAGGTACT
  			TCTACTGAACCGTCCGAGGGCA
  			GCGCACCAGGTAGCCCTGCTGG
  			CTCTCCAACCTCCACCGAAGAA
  			GGTACCTCTGAAAGCGCAACCC
  			CTGAATCCGGCCCAGGTAGCGA
  			ACCGGCAACCTCCGGTTCTGAA
  			ACCCCAGGTACTTCTGAAAGCG
  			CTACTCCTGAGTCCGGCCCAGG
  			TAGCCCGGCTGGCTCTCCGACT
  			TCCACCGAGGAAGGTAGCCCGG
  			CTGGCTCTCCAACTTCTACTGA
  			AGAAGGTACTTCTACCGAACCT
  			TCCGAGGGCAGCGCACCAGGTA
  			CTTCTGAAAGCGCTACCCCTGA
  			GTCCGGCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAATCCGGTC
  			CAGGTACTTCTGAAAGCGCTAC
  			CCCGGAATCTGGCCCAGGTAGC
  			GAACCGGCTACTTCTGGTTCTG
  			AAACCCCAGGTAGCGAACCGGC
  			TACCTCCGGTTCTGAAACTCCA
  			GGTAGCCCAGCAGGCTCTCCGA
  			CTTCCACTGAGGAAGGTACTTC
  			TACTGAACCTTCCGAAGGCAGC
  			GCACCAGGTACCTCTACTGAAC
  			CTTCTGAGGGCAGCGCTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	718	GCCAACGCGTTCCTGGAGGAGC	719
  Kallikrein-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE288	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	SPFRSTGGGGTSES		AGGTCATCATCCCCAGCACGTA
  	ATPESGPGSEPATS		CGTCCCGGGCACCACCAACCAC
  	GSETPGTSESATPE		GACATCGCGCTGCTCCGCCTGC
  	SGPGSEPATSGSET		ACCAGCCCGTGGTCCTCACTGA
  	PGTSESATPESGPG		CCATGTGGTGCCCCTCTGCCTG
  	TSTEPSEGSAPGSP		CCCGAACGGACGTTCTCTGAGA
  	AGSPTSTEEGTSES		GGACGCTGGCCTTCGTGCGCTT
  	ATPESGPGSEPATS		CTCATTGGTCAGCGGCTGGGGC
  	GSETPGTSESATPE		CAGCTGCTGGACCGTGGCGCCA
  	SGPGSPAGSPTSTE		CGGCCCTGGAGCTCATGGTCCT
  	EGSPAGSPTSTEEG		CAACGTGCCCCGGCTGATGACC
  	TSTEPSEGSAPGTS		CAGGACTGCCTGCAGCAGTCAC
  	ESATPESGPGTSES		GGAAGGTGGGAGACTCCCCAA
  	ATPESGPGTSESAT		ATATCACGGAGTACATGTTCTG
  	PESGPGSEPATSGS		TGCCGGCTACTCGGATGGCAGC
  	ETPGSEPATSGSET		AAGGACTCCTGCAAGGGGGAC
  	PGSPAGSPTSTEEG		AGTGGAGGCCCACATGCCACCC
  	TSTEPSEGSAPGTS		ACTACCGGGGCACGTGGTACCT
  	TEPSEGSAPGSEPA		GACGGGCATCGTCAGCTGGGGC
  	TSGSETPGTSESAT		CAGGGCTGCGCAACCGTGGGCC
  	PESGPGTSTEPSEG		ACTTTGGGGTGTACACCAGGGT
  	SAP		CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-	ANAFLEELRPGSLE	720	GCCAACGCGTTCCTGGAGGAGC	721
  Kallikrein-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE864	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	SPFRSTGGGGSPA		AGGTCATCATCCCCAGCACGTA
  	GSPTSTEEGTSESA		CGTCCCGGGCACCACCAACCAC
  	TPESGPGTSTEPSE		GACATCGCGCTGCTCCGCCTGC
  	GSAPGSPAGSPTST		ACCAGCCCGTGGTCCTCACTGA
  	EEGTSTEPSEGSAP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGT		CCCGAACGGACGTTCTCTGAGA
  	SESATPESGPGSEP		GGACGCTGGCCTTCGTGCGCTT
  	ATSGSETPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGSPAGSPT		CAGCTGCTGGACCGTGGCGCCA
  	STEEGTSESATPES		CGGCCCTGGAGCTCATGGTCCT
  	GPGTSTEPSEGSAP		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGS		CAGGACTGCCTGCAGCAGTCAC
  	PAGSPTSTEEGTST		GGAAGGTGGGAGACTCCCCAA
  	EPSEGSAPGTSTEP		ATATCACGGAGTACATGTTCTG
  	SEGSAPGTSESATP		TGCCGGCTACTCGGATGGCAGC
  	ESGPGTSTEPSEGS		AAGGACTCCTGCAAGGGGGAC
  	APGTSESATPESGP		AGTGGAGGCCCACATGCCACCC
  	GSEPATSGSETPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGTST		GACGGGCATCGTCAGCTGGGGC
  	EPSEGSAPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSESATP		ACTTTGGGGTGTACACCAGGGT
  	ESGPGSPAGSPTST		CTCCCAGTACATCGAGTGGCTG
  	EEGTSESATPESGP		CAAAAGCTCATGCGCTCAGAGC
  	GSEPATSGSETPGT		CACGCCCAGGAGTCCTCCTGCG
  	SESATPESGPGTST		AGCCCCATTTCCCGGT0GGTGGT
  	EPSEGSAPGTSTEP		AGCCCGGCTGGCTCTCCTACCT
  	SEGSAPGTSTEPSE		CTACTGAGGAAGGTACTTCTGA
  	GSAPGTSTEPSEGS		AAGCGCTACTCCTGAGTCTGGT
  	APGTSTEPSEGSAP		CCAGGTACCTCTACTGAACCGT
  	GTSTEPSEGSAPGS		CCGAAGGTAGCGCTCCAGGTAG
  	PAGSPTSTEEGTST		CCCAGCAGGCTCTCCGACTTCC
  	EPSEGSAPGTSESA		ACTGAGGAAGGTACTTCTACTG
  	TPESGPGSEPATSG		AACCTTCCGAAGGCAGCGCACC
  	SETPGTSESATPES		AGGTACCTCTACTGAACCTTCT
  	GPGSEPATSGSETP		GAGGGCAGCGCTCCAGGTACTT
  	GTSESATPESGPGT		CTGAAAGCGCTACCCCGGAATC
  	STEPSEGSAPGTSE		TGGCCCAGGTAGCGAACCGGCT
  	SATPESGPGSPAGS		ACTTCTGGTTCTGAAACCCCAG
  	PTSTEEGSPAGSPT		GTAGCGAACCGGCTACCTCCGG
  	STEEGSPAGSPTST		TTCTGAAACTCCAGGTAGCCCG
  	EEGTSESATPESGP		GCAGGCTCTCCGACCTCTACTG
  	GTSTEPSEGSAPGT		AGGAAGGTACTTCTGAAAGCGC
  	SESATPESGPGSEP		AACCCCGGAGTCCGGCCCAGGT
  	ATSGSETPGTSESA		ACCTCTACCGAACCGTCTGAGG
  	TPESGPGSEPATSG		GCAGCGCACCAGGTACTTCTAC
  	SETPGTSESATPES		CGAACCGTCCGAGGGTAGCGCA
  	GPGTSTEPSEGSAP		CCAGGTAGCCCAGCAGGTTCTC
  	GSPAGSPTSTEEGT		CTACCTCCACCGAGGAAGGTAC
  	SESATPESGPGSEP		TTCTACCGAACCGTCCGAGGGT
  	ATSGSETPGTSESA		AGCGCACCAGGTACCTCTACTG
  	TPESGPGSPAGSPT		AACCTTCTGAGGGCAGCGCTCC
  	STEEGSPAGSPTST		AGGTACTTCTGAAAGCGCTACC
  	EEGTSTEPSEGSAP		CCGGAGTCCGGTCCAGGTACTT
  	GTSESATPESGPGT		CTACTGAACCGTCCGAAGGTAG
  	SESATPESGPGTSE		CGCACCAGGTACTTCTGAAAGC
  	SATPESGPGSEPAT		GCAACCCCTGAATCCGGTCCAG
  	SGSETPGSEPATSG		GTAGCGAACCGGCTACTTCTGG
  	SETPGSPAGSPTST		CTCTGAGACTCCAGGTACTTCT
  	EEGTSTEPSEGSAP		ACCGAACCGTCCGAAGGTAGCG
  	GTSTEPSEGSAPGS		CACCAGGTACTTCTACTGAACC
  	EPATSGSETPGTSE		GTCTGAAGGTAGCGCACCAGGT
  	SATPESGPGTSTEP		ACTTCTGAAAGCGCAACCCCGG
  	SEGSAP		AATCCGGCCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAGTCCGGC
  			CCAGGTAGCCCTGCTGGCTCTC
  			CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	722	TATAATTCAGGTAAATTGGAAG	723
  Kallikrein-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE288	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGS		AATTACAGTTGTCGCAGGTGAA
  	PFRSTGGGGTSESA		CATAATATTGAGGAGACAGAAC
  	TPESGPGSEPATSG		ATACAGAGCAAAAGCGAAATG
  	SETPGTSESATPES		TGATTCGAATTATTCCTCACCAC
  	GPGSEPATSGSETP		AACTACAATGCAGCTATTAATA
  	GTSESATPESGPGT		AGTACAACCATGACATTGCCCT
  	STEPSEGSAPGSPA		TCTGGAACTGGACGAACCCTTA
  	GSPTSTEEGTSESA		GTGCTAAACAGCTACGTTACAC
  	TPESGPGSEPATSG		CTATTTGCATTGCTGACAAGGA
  	SETPGTSESATPES		ATACACGAACATCTTCCTCAAA
  	GPGSPAGSPTSTEE		TTTGGATCTGGCTATGTAAGTG
  	GSPAGSPTSTEEGT		GCTGGGGAAGAGTCTTCCACAA
  	STEPSEGSAPGTSE		AGGGAGATCAGCTTTAGTTCTT
  	SATPESGPGTSESA		CAGTACCTTAGAGTTCCACTTG
  	TPESGPGTSESATP		TTGACCGAGCCACATGTCTTCG
  	ESGPGSEPATSGSE		ATCTACAAAGTTCACCATCTAT
  	TPGSEPATSGSETP		AACAACATGTTCTGTGCTGGCT
  	GSPAGSPTSTEEGT		TCCATGAAGGAGGTAGAGATTC
  	STEPSEGSAPGTST		ATGTCAAGGAGATAGTGGGGG
  	EPSEGSAPGSEPAT		ACCCCATGTTACTGAAGTGGAA
  	SGSETPGTSESATP		GGGACCAGTTTCTTAACTGGAA
  	ESGPGTSTEPSEGS		TTATTAGCTGGGGTGAAGAGTG
  	AP		TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0GGT
  			GGTACCTCTGAAAGCGCAACTC
  			CTGAGTCTGGCCCAGGTAGCGA
  			ACCTGCTACCTCCGGCTCTGAG
  			ACTCCAGGTACCTCTGAAAGCG
  			CAACCCCGGAATCTGGTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCAGGTA
  			GCCCTGCTGGCTCTCCAACCTC
  			CACCGAAGAAGGTACCTCTGAA
  			AGCGCAACCCCTGAATCCGGCC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACCCCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTACTTC
  			TACCGAACCTTCCGAGGGCAGC
  			GCACCAGGTACTTCTGAAAGCG
  			CTACCCCTGAGTCCGGCCCAGG
  			TACTTCTGAAAGCGCTACTCCT
  			GAATCCGGTCCAGGTACTTCTG
  			AAAGCGCTACCCCGGAATCTGG
  			CCCAGGTAGCGAACCGGCTACT
  			TCTGGTTCTGAAACCCCAGGTA
  			GCGAACCGGCTACCTCCGGTTC
  			TGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGG
  			AAGGTACTTCTACTGAACCTTC
  			CGAAGGCAGCGCACCAGGTACC
  			TCTACTGAACCTTCTGAGGGCA
  			GCGCTCCAGGTAGCGAACCTGC
  			AACCTCTGGCTCTGAAACCCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CTGAATCTGGCCCAGGTACTTC
  			TACTGAACCGTCCGAGGGCAGC
  			GCACCA
  FIX-	YNSGKLEEFVQGN	724	TATAATTCAGGTAAATTGGAAG	725
  Kallikrein-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE864	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGS		AATTACAGTTGTCGCAGGTGAA
  	PFRSTGGGGSPAG		CATAATATTGAGGAGACAGAAC
  	SPTSTEEGTSESAT		ATACAGAGCAAAAGCGAAATG
  	PESGPGTSTEPSEG		TGATTCGAATTATTCCTCACCAC
  	SAPGSPAGSPTSTE		AACTACAATGCAGCTATTAATA
  	EGTSTEPSEGSAPG		AGTACAACCATGACATTGCCCT
  	TSTEPSEGSAPGTS		TCTGGAACTGGACGAACCCTTA
  	ESATPESGPGSEPA		GTGCTAAACAGCTACGTTACAC
  	TSGSETPGSEPATS		CTATTTGCATTGCTGACAAGGA
  	GSETPGSPAGSPTS		ATACACGAACATCTTCCTCAAA
  	TEEGTSESATPESG		TTTGGATCTGGCTATGTAAGTG
  	PGTSTEPSEGSAPG		GCTGGGGAAGAGTCTTCCACAA
  	TSTEPSEGSAPGSP		AGGGAGATCAGCTTTAGTTCTT
  	AGSPTSTEEGTSTE		CAGTACCTTAGAGTTCCACTTG
  	PSEGSAPGTSTEPS		TTGACCGAGCCACATGTCTTCG
  	EGSAPGTSESATPE		ATCTACAAAGTTCACCATCTAT
  	SGPGTSTEPSEGSA		AACAACATGTTCTGTGCTGGCT
  	PGTSESATPESGPG		TCCATGAAGGAGGTAGAGATTC
  	SEPATSGSETPGTS		ATGTCAAGGAGATAGTGGGGG
  	TEPSEGSAPGTSTE		ACCCCATGTTACTGAAGTGGAA
  	PSEGSAPGTSESAT		GGGACCAGTTTCTTAACTGGAA
  	PESGPGTSESATPE		TTATTAGCTGGGGTGAAGAGTG
  	SGPGSPAGSPTSTE		TGCAATGAAAGGCAAATATGGA
  	EGTSESATPESGPG		ATATATACCAAGGTATCCCGGT
  	SEPATSGSETPGTS		ATGTCAACTGGATTAAGGAAAA
  	ESATPESGPGTSTE		AACAAAGCTCACTGGGGT0GGT
  	PSEGSAPGTSTEPS		GGTAGCCCGGCTGGCTCTCCTA
  	EGSAPGTSTEPSEG		CCTCTACTGAGGAAGGTACTTC
  	SAPGTSTEPSEGSA		TGAAAGCGCTACTCCTGAGTCT
  	PGTSTEPSEGSAPG		GGTCCAGGTACCTCTACTGAAC
  	TSTEPSEGSAPGSP		CGTCCGAAGGTAGCGCTCCAGG
  	AGSPTSTEEGTSTE		TAGCCCAGCAGGCTCTCCGACT
  	PSEGSAPGTSESAT		TCCACTGAGGAAGGTACTTCTA
  	PESGPGSEPATSGS		CTGAACCTTCCGAAGGCAGCGC
  	ETPGTSESATPESG		ACCAGGTACCTCTACTGAACCT
  	PGSEPATSGSETPG		TCTGAGGGCAGCGCTCCAGGTA
  	TSESATPESGPGTS		CTTCTGAAAGCGCTACCCCGGA
  	TEPSEGSAPGTSES		ATCTGGCCCAGGTAGCGAACCG
  	ATPESGPGSPAGSP		GCTACTTCTGGTTCTGAAACCC
  	TSTEEGSPAGSPTS		CAGGTAGCGAACCGGCTACCTC
  	TEEGSPAGSPTSTE		CGGTTCTGAAACTCCAGGTAGC
  	EGTSESATPESGPG		CCGGCAGGCTCTCCGACCTCTA
  	TSTEPSEGSAPGTS		CTGAGGAAGGTACTTCTGAAAG
  	ESATPESGPGSEPA		CGCAACCCCGGAGTCCGGCCCA
  	TSGSETPGTSESAT		GGTACCTCTACCGAACCGTCTG
  	PESGPGSEPATSGS		AGGGCAGCGCACCAGGTACTTC
  	ETPGTSESATPESG		TACCGAACCGTCCGAGGGTAGC
  	PGTSTEPSEGSAPG		GCACCAGGTAGCCCAGCAGGTT
  	SPAGSPTSTEEGTS		CTCCTACCTCCACCGAGGAAGG
  	ESATPESGPGSEPA		TACTTCTACCGAACCGTCCGAG
  	TSGSETPGTSESAT		GGTAGCGCACCAGGTACCTCTA
  	PESGPGSPAGSPTS		CTGAACCTTCTGAGGGCAGCGC
  	TEEGSPAGSPTSTE		TCCAGGTACTTCTGAAAGCGCT
  	EGTSTEPSEGSAPG		ACCCCGGAGTCCGGTCCAGGTA
  	TSESATPESGPGTS		CTTCTACTGAACCGTCCGAAGG
  	ESATPESGPGTSES		TAGCGCACCAGGTACTTCTGAA
  	ATPESGPGSEPATS		AGCGCAACCCCTGAATCCGGTC
  	GSETPGSEPATSGS		CAGGTAGCGAACCGGCTACTTC
  	ETPGSPAGSPTSTE		TGGCTCTGAGACTCCAGGTACT
  	EGTSTEPSEGSAPG		TCTACCGAACCGTCCGAAGGTA
  	TSTEPSEGSAPGSE		GCGCACCAGGTACTTCTACTGA
  	PATSGSETPGTSES		ACCGTCTGAAGGTAGCGCACCA
  	ATPESGPGTSTEPS		GGTACTTCTGAAAGCGCAACCC
  	EGSAP		CGGAATCCGGCCCAGGTACCTC
  			TGAAAGCGCAACCCCGGAGTCC
  			GGCCCAGGTAGCCCTGCTGGCT
  			CTCCAACCTCCACCGAAGAAGG
  			TACCTCTGAAAGCGCAACCCCT
  			GAATCCGGCCCAGGTAGCGAAC
  			CGGCAACCTCCGGTTCTGAAAC
  			CCCAGGTACCTCTGAAAGCGCT
  			ACTCCGGAGTCTGGCCCAGGTA
  			CCTCTACTGAACCGTCTGAGGG
  			TAGCGCTCCAGGTACTTCTACT
  			GAACCGTCCGAAGGTAGCGCAC
  			CAGGTACTTCTACCGAACCGTC
  			CGAAGGCAGCGCTCCAGGTACC
  			TCTACTGAACCTTCCGAGGGCA
  			GCGCTCCAGGTACCTCTACCGA
  			ACCTTCTGAAGGTAGCGCACCA
  			GGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTAGCCC
  			AGCAGGTTCTCCTACCTCCACC
  			GAGGAAGGTACTTCTACCGAAC
  			CGTCCGAGGGTAGCGCACCAGG
  			TACCTCTGAAAGCGCAACTCCT
  			GAGTCTGGCCCAGGTAGCGAAC
  			CTGCTACCTCCGGCTCTGAGAC
  			TCCAGGTACCTCTGAAAGCGCA
  			ACCCCGGAATCTGGTCCAGGTA
  			GCGAACCTGCAACCTCTGGCTC
  			TGAAACCCCAGGTACCTCTGAA
  			AGCGCTACTCCTGAATCTGGCC
  			CAGGTACTTCTACTGAACCGTC
  			CGAGGGCAGCGCACCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTAGCCC
  			GGCAGGCTCTCCGACCTCTACT
  			GAGGAAGGTACTTCTGAAAGCG
  			CAACCCCGGAGTCCGGCCCAGG
  			TACCTCTACCGAACCGTCTGAG
  			GGCAGCGCACCAGGTACCTCTG
  			AAAGCGCAACTCCTGAGTCTGG
  			CCCAGGTAGCGAACCTGCTACC
  			TCCGGCTCTGAGACTCCAGGTA
  			CCTCTGAAAGCGCAACCCCGGA
  			ATCTGGTCCAGGTAGCGAACCT
  			GCAACCTCTGGCTCTGAAACCC
  			CAGGTACCTCTGAAAGCGCTAC
  			TCCTGAATCTGGCCCAGGTACT
  			TCTACTGAACCGTCCGAGGGCA
  			GCGCACCAGGTAGCCCTGCTGG
  			CTCTCCAACCTCCACCGAAGAA
  			GGTACCTCTGAAAGCGCAACCC
  			CTGAATCCGGCCCAGGTAGCGA
  			ACCGGCAACCTCCGGTTCTGAA
  			ACCCCAGGTACTTCTGAAAGCG
  			CTACTCCTGAGTCCGGCCCAGG
  			TAGCCCGGCTGGCTCTCCGACT
  			TCCACCGAGGAAGGTAGCCCGG
  			CTGGCTCTCCAACTTCTACTGA
  			AGAAGGTACTTCTACCGAACCT
  			TCCGAGGGCAGCGCACCAGGTA
  			CTTCTGAAAGCGCTACCCCTGA
  			GTCCGGCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAATCCGGTC
  			CAGGTACTTCTGAAAGCGCTAC
  			CCCGGAATCTGGCCCAGGTAGC
  			GAACCGGCTACTTCTGGTTCTG
  			AAACCCCAGGTAGCGAACCGGC
  			TACCTCCGGTTCTGAAACTCCA
  			GGTAGCCCAGCAGGCTCTCCGA
  			CTTCCACTGAGGAAGGTACTTC
  			TACTGAACCTTCCGAAGGCAGC
  			GCACCAGGTACCTCTACTGAAC
  			CTTCTGAGGGCAGCGCTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	726	GCCAACGCGTTCCTGGAGGAGC	727
  FIIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE288	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	LTPRSLLVGGTSES		AGGTCATCATCCCCAGCACGTA
  	ATPESGPGSEPATS		CGTCCCGGGCACCACCAACCAC
  	GSETPGTSESATPE		GACATCGCGCTGCTCCGCCTGC
  	SGPGSEPATSGSET		ACCAGCCCGTGGTCCTCACTGA
  	PGTSESATPESGPG		CCATGTGGTGCCCCTCTGCCTG
  	TSTEPSEGSAPGSP		CCCGAACGGACGTTCTCTGAGA
  	AGSPTSTEEGTSES		GGACGCTGGCCTTCGTGCGCTT
  	ATPESGPGSEPATS		CTCATTGGTCAGCGGCTGGGGC
  	GSETPGTSESATPE		CAGCTGCTGGACCGTGGCGCCA
  	SGPGSPAGSPTSTE		CGGCCCTGGAGCTCATGGTCCT
  	EGSPAGSPTSTEEG		CAACGTGCCCCGGCTGATGACC
  	TSTEPSEGSAPGTS		CAGGACTGCCTGCAGCAGTCAC
  	ESATPESGPGTSES		GGAAGGTGGGAGACTCCCCAA
  	ATPESGPGTSESAT		ATATCACGGAGTACATGTTCTG
  	PESGPGSEPATSGS		TGCCGGCTACTCGGATGGCAGC
  	ETPGSEPATSGSET		AAGGACTCCTGCAAGGGGGAC
  	PGSPAGSPTSTEEG		AGTGGAGGCCCACATGCCACCC
  	TSTEPSEGSAPGTS		ACTACCGGGGCACGTGGTACCT
  	TEPSEGSAPGSEPA		GACGGGCATCGTCAGCTGGGGC
  	TSGSETPGTSESAT		CAGGGCTGCGCAACCGTGGGCC
  	PESGPGTSTEPSEG		ACTTTGGGGTGTACACCAGGGT
  	SAP		CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-	ANAFLEELRPGSLE	728	GCCAACGCGTTCCTGGAGGAGC	729
  FIIa-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE864	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	LTPRSLLVGGSPA		AGGTCATCATCCCCAGCACGTA
  	GSPTSTEEGTSESA		CGTCCCGGGCACCACCAACCAC
  	TPESGPGTSTEPSE		GACATCGCGCTGCTCCGCCTGC
  	GSAPGSPAGSPTST		ACCAGCCCGTGGTCCTCACTGA
  	EEGTSTEPSEGSAP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGT		CCCGAACGGACGTTCTCTGAGA
  	SESATPESGPGSEP		GGACGCTGGCCTTCGTGCGCTT
  	ATSGSETPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGSPAGSPT		CAGCTGCTGGACCGTGGCGCCA
  	STEEGTSESATPES		CGGCCCTGGAGCTCATGGTCCT
  	GPGTSTEPSEGSAP		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGS		CAGGACTGCCTGCAGCAGTCAC
  	PAGSPTSTEEGTST		GGAAGGTGGGAGACTCCCCAA
  	EPSEGSAPGTSTEP		ATATCACGGAGTACATGTTCTG
  	SEGSAPGTSESATP		TGCCGGCTACTCGGATGGCAGC
  	ESGPGTSTEPSEGS		AAGGACTCCTGCAAGGGGGAC
  	APGTSESATPESGP		AGTGGAGGCCCACATGCCACCC
  	GSEPATSGSETPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGTST		GACGGGCATCGTCAGCTGGGGC
  	EPSEGSAPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSESATP		ACTTTGGGGTGTACACCAGGGT
  	ESGPGSPAGSPTST		CTCCCAGTACATCGAGTGGCTG
  	EEGTSESATPESGP		CAAAAGCTCATGCGCTCAGAGC
  	GSEPATSGSETPGT		CACGCCCAGGAGTCCTCCTGCG
  	SESATPESGPGTST		AGCCCCATTTCCCGGT0GGTGGT
  	EPSEGSAPGTSTEP		AGCCCGGCTGGCTCTCCTACCT
  	SEGSAPGTSTEPSE		CTACTGAGGAAGGTACTTCTGA
  	GSAPGTSTEPSEGS		AAGCGCTACTCCTGAGTCTGGT
  	APGTSTEPSEGSAP		CCAGGTACCTCTACTGAACCGT
  	GTSTEPSEGSAPGS		CCGAAGGTAGCGCTCCAGGTAG
  	PAGSPTSTEEGTST		CCCAGCAGGCTCTCCGACTTCC
  	EPSEGSAPGTSESA		ACTGAGGAAGGTACTTCTACTG
  	TPESGPGSEPATSG		AACCTTCCGAAGGCAGCGCACC
  	SETPGTSESATPES		AGGTACCTCTACTGAACCTTCT
  	GPGSEPATSGSETP		GAGGGCAGCGCTCCAGGTACTT
  	GTSESATPESGPGT		CTGAAAGCGCTACCCCGGAATC
  	STEPSEGSAPGTSE		TGGCCCAGGTAGCGAACCGGCT
  	SATPESGPGSPAGS		ACTTCTGGTTCTGAAACCCCAG
  	PTSTEEGSPAGSPT		GTAGCGAACCGGCTACCTCCGG
  	STEEGSPAGSPTST		TTCTGAAACTCCAGGTAGCCCG
  	EEGTSESATPESGP		GCAGGCTCTCCGACCTCTACTG
  	GTSTEPSEGSAPGT		AGGAAGGTACTTCTGAAAGCGC
  	SESATPESGPGSEP		AACCCCGGAGTCCGGCCCAGGT
  	ATSGSETPGTSESA		ACCTCTACCGAACCGTCTGAGG
  	TPESGPGSEPATSG		GCAGCGCACCAGGTACTTCTAC
  	SETPGTSESATPES		CGAACCGTCCGAGGGTAGCGCA
  	GPGTSTEPSEGSAP		CCAGGTAGCCCAGCAGGTTCTC
  	GSPAGSPTSTEEGT		CTACCTCCACCGAGGAAGGTAC
  	SESATPESGPGSEP		TTCTACCGAACCGTCCGAGGGT
  	ATSGSETPGTSESA		AGCGCACCAGGTACCTCTACTG
  	TPESGPGSPAGSPT		AACCTTCTGAGGGCAGCGCTCC
  	STEEGSPAGSPTST		AGGTACTTCTGAAAGCGCTACC
  	EEGTSTEPSEGSAP		CCGGAGTCCGGTCCAGGTACTT
  	GTSESATPESGPGT		CTACTGAACCGTCCGAAGGTAG
  	SESATPESGPGTSE		CGCACCAGGTACTTCTGAAAGC
  	SATPESGPGSEPAT		GCAACCCCTGAATCCGGTCCAG
  	SGSETPGSEPATSG		GTAGCGAACCGGCTACTTCTGG
  	SETPGSPAGSPTST		CTCTGAGACTCCAGGTACTTCT
  	EEGTSTEPSEGSAP		ACCGAACCGTCCGAAGGTAGCG
  	GTSTEPSEGSAPGS		CACCAGGTACTTCTACTGAACC
  	EPATSGSETPGTSE		GTCTGAAGGTAGCGCACCAGGT
  	SATPESGPGTSTEP		ACTTCTGAAAGCGCAACCCCGG
  	SEGSAP		AATCCGGCCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAGTCCGGC
  			CCAGGTAGCCCTGCTGGCTCTC
  			CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	730	TATAATTCAGGTAAATTGGAAG	731
  FIIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE288	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGL		AATTACAGTTGTCGCAGGTGAA
  	TPRSLLVGGTSESA		CATAATATTGAGGAGACAGAAC
  	TPESGPGSEPATSG		ATACAGAGCAAAAGCGAAATG
  	SETPGTSESATPES		TGATTCGAATTATTCCTCACCAC
  	GPGSEPATSGSETP		AACTACAATGCAGCTATTAATA
  	GTSESATPESGPGT		AGTACAACCATGACATTGCCCT
  	STEPSEGSAPGSPA		TCTGGAACTGGACGAACCCTTA
  	GSPTSTEEGTSESA		GTGCTAAACAGCTACGTTACAC
  	TPESGPGSEPATSG		CTATTTGCATTGCTGACAAGGA
  	SETPGTSESATPES		ATACACGAACATCTTCCTCAAA
  	GPGSPAGSPTSTEE		TTTGGATCTGGCTATGTAAGTG
  	GSPAGSPTSTEEGT		GCTGGGGAAGAGTCTTCCACAA
  	STEPSEGSAPGTSE		AGGGAGATCAGCTTTAGTTCTT
  	SATPESGPGTSESA		CAGTACCTTAGAGTTCCACTTG
  	TPESGPGTSESATP		TTGACCGAGCCACATGTCTTCG
  	ESGPGSEPATSGSE		ATCTACAAAGTTCACCATCTAT
  	TPGSEPATSGSETP		AACAACATGTTCTGTGCTGGCT
  	GSPAGSPTSTEEGT		TCCATGAAGGAGGTAGAGATTC
  	STEPSEGSAPGTST		ATGTCAAGGAGATAGTGGGGG
  	EPSEGSAPGSEPAT		ACCCCATGTTACTGAAGTGGAA
  	SGSETPGTSESATP		GGGACCAGTTTCTTAACTGGAA
  	ESGPGTSTEPSEGS		TTATTAGCTGGGGTGAAGAGTG
  	AP		TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0GGT
  			GGTACCTCTGAAAGCGCAACTC
  			CTGAGTCTGGCCCAGGTAGCGA
  			ACCTGCTACCTCCGGCTCTGAG
  			ACTCCAGGTACCTCTGAAAGCG
  			CAACCCCGGAATCTGGTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCAGGTA
  			GCCCTGCTGGCTCTCCAACCTC
  			CACCGAAGAAGGTACCTCTGAA
  			AGCGCAACCCCTGAATCCGGCC
  			CAGGTAGCGAACCGGCAACCTC
  			CGGTTCTGAAACCCCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTACTTC
  			TACCGAACCTTCCGAGGGCAGC
  			GCACCAGGTACTTCTGAAAGCG
  			CTACCCCTGAGTCCGGCCCAGG
  			TACTTCTGAAAGCGCTACTCCT
  			GAATCCGGTCCAGGTACTTCTG
  			AAAGCGCTACCCCGGAATCTGG
  			CCCAGGTAGCGAACCGGCTACT
  			TCTGGTTCTGAAACCCCAGGTA
  			GCGAACCGGCTACCTCCGGTTC
  			TGAAACTCCAGGTAGCCCAGCA
  			GGCTCTCCGACTTCCACTGAGG
  			AAGGTACTTCTACTGAACCTTC
  			CGAAGGCAGCGCACCAGGTACC
  			TCTACTGAACCTTCTGAGGGCA
  			GCGCTCCAGGTAGCGAACCTGC
  			AACCTCTGGCTCTGAAACCCCA
  			GGTACCTCTGAAAGCGCTACTC
  			CTGAATCTGGCCCAGGTACTTC
  			TACTGAACCGTCCGAGGGCAGC
  			GCACCA
  FIX-	YNSGKLEEFVQGN	732	TATAATTCAGGTAAATTGGAAG	733
  FIIa-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  AE864	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGL		AATTACAGTTGTCGCAGGTGAA
  	TPRSLLVGGSPAGS		CATAATATTGAGGAGACAGAAC
  	PTSTEEGTSESATP		ATACAGAGCAAAAGCGAAATG
  	ESGPGTSTEPSEGS		TGATTCGAATTATTCCTCACCAC
  	APGSPAGSPTSTEE		AACTACAATGCAGCTATTAATA
  	GTSTEPSEGSAPGT		AGTACAACCATGACATTGCCCT
  	STEPSEGSAPGTSE		TCTGGAACTGGACGAACCCTTA
  	SATPESGPGSEPAT		GTGCTAAACAGCTACGTTACAC
  	SGSETPGSEPATSG		CTATTTGCATTGCTGACAAGGA
  	SETPGSPAGSPTST		ATACACGAACATCTTCCTCAAA
  	EEGTSESATPESGP		TTTGGATCTGGCTATGTAAGTG
  	GTSTEPSEGSAPGT		GCTGGGGAAGAGTCTTCCACAA
  	STEPSEGSAPGSPA		AGGGAGATCAGCTTTAGTTCTT
  	GSPTSTEEGTSTEP		CAGTACCTTAGAGTTCCACTTG
  	SEGSAPGTSTEPSE		TTGACCGAGCCACATGTCTTCG
  	GSAPGTSESATPES		ATCTACAAAGTTCACCATCTAT
  	GPGTSTEPSEGSAP		AACAACATGTTCTGTGCTGGCT
  	GTSESATPESGPGS		TCCATGAAGGAGGTAGAGATTC
  	EPATSGSETPGTST		ATGTCAAGGAGATAGTGGGGG
  	EPSEGSAPGTSTEP		ACCCCATGTTACTGAAGTGGAA
  	SEGSAPGTSESATP		GGGACCAGTTTCTTAACTGGAA
  	ESGPGTSESATPES		TTATTAGCTGGGGTGAAGAGTG
  	GPGSPAGSPTSTEE		TGCAATGAAAGGCAAATATGGA
  	GTSESATPESGPGS		ATATATACCAAGGTATCCCGGT
  	EPATSGSETPGTSE		ATGTCAACTGGATTAAGGAAAA
  	SATPESGPGTSTEP		AACAAAGCTCACTGGGGT0GGT
  	SEGSAPGTSTEPSE		GGTAGCCCGGCTGGCTCTCCTA
  	GSAPGTSTEPSEGS		CCTCTACTGAGGAAGGTACTTC
  	APGTSTEPSEGSAP		TGAAAGCGCTACTCCTGAGTCT
  	GTSTEPSEGSAPGT		GGTCCAGGTACCTCTACTGAAC
  	STEPSEGSAPGSPA		CGTCCGAAGGTAGCGCTCCAGG
  	GSPTSTEEGTSTEP		TAGCCCAGCAGGCTCTCCGACT
  	SEGSAPGTSESATP		TCCACTGAGGAAGGTACTTCTA
  	ESGPGSEPATSGSE		CTGAACCTTCCGAAGGCAGCGC
  	TPGTSESATPESGP		ACCAGGTACCTCTACTGAACCT
  	GSEPATSGSETPGT		TCTGAGGGCAGCGCTCCAGGTA
  	SESATPESGPGTST		CTTCTGAAAGCGCTACCCCGGA
  	EPSEGSAPGTSESA		ATCTGGCCCAGGTAGCGAACCG
  	TPESGPGSPAGSPT		GCTACTTCTGGTTCTGAAACCC
  	STEEGSPAGSPTST		CAGGTAGCGAACCGGCTACCTC
  	EEGSPAGSPTSTEE		CGGTTCTGAAACTCCAGGTAGC
  	GTSESATPESGPGT		CCGGCAGGCTCTCCGACCTCTA
  	STEPSEGSAPGTSE		CTGAGGAAGGTACTTCTGAAAG
  	SATPESGPGSEPAT		CGCAACCCCGGAGTCCGGCCCA
  	SGSETPGTSESATP		GGTACCTCTACCGAACCGTCTG
  	ESGPGSEPATSGSE		AGGGCAGCGCACCAGGTACTTC
  	TPGTSESATPESGP		TACCGAACCGTCCGAGGGTAGC
  	GTSTEPSEGSAPGS		GCACCAGGTAGCCCAGCAGGTT
  	PAGSPTSTEEGTSE		CTCCTACCTCCACCGAGGAAGG
  	SATPESGPGSEPAT		TACTTCTACCGAACCGTCCGAG
  	SGSETPGTSESATP		GGTAGCGCACCAGGTACCTCTA
  	ESGPGSPAGSPTST		CTGAACCTTCTGAGGGCAGCGC
  	EEGSPAGSPTSTEE		TCCAGGTACTTCTGAAAGCGCT
  	GTSTEPSEGSAPGT		ACCCCGGAGTCCGGTCCAGGTA
  	SESATPESGPGTSE		CTTCTACTGAACCGTCCGAAGG
  	SATPESGPGTSESA		TAGCGCACCAGGTACTTCTGAA
  	TPESGPGSEPATSG		AGCGCAACCCCTGAATCCGGTC
  	SETPGSEPATSGSE		CAGGTAGCGAACCGGCTACTTC
  	TPGSPAGSPTSTEE		TGGCTCTGAGACTCCAGGTACT
  	GTSTEPSEGSAPGT		TCTACCGAACCGTCCGAAGGTA
  	STEPSEGSAPGSEP		GCGCACCAGGTACTTCTACTGA
  	ATSGSETPGTSESA		ACCGTCTGAAGGTAGCGCACCA
  	TPESGPGTSTEPSE		GGTACTTCTGAAAGCGCAACCC
  	GSAP		CGGAATCCGGCCCAGGTACCTC
  			TGAAAGCGCAACCCCGGAGTCC
  			GGCCCAGGTAGCCCTGCTGGCT
  			CTCCAACCTCCACCGAAGAAGG
  			TACCTCTGAAAGCGCAACCCCT
  			GAATCCGGCCCAGGTAGCGAAC
  			CGGCAACCTCCGGTTCTGAAAC
  			CCCAGGTACCTCTGAAAGCGCT
  			ACTCCGGAGTCTGGCCCAGGTA
  			CCTCTACTGAACCGTCTGAGGG
  			TAGCGCTCCAGGTACTTCTACT
  			GAACCGTCCGAAGGTAGCGCAC
  			CAGGTACTTCTACCGAACCGTC
  			CGAAGGCAGCGCTCCAGGTACC
  			TCTACTGAACCTTCCGAGGGCA
  			GCGCTCCAGGTACCTCTACCGA
  			ACCTTCTGAAGGTAGCGCACCA
  			GGTACTTCTACCGAACCGTCCG
  			AGGGTAGCGCACCAGGTAGCCC
  			AGCAGGTTCTCCTACCTCCACC
  			GAGGAAGGTACTTCTACCGAAC
  			CGTCCGAGGGTAGCGCACCAGG
  			TACCTCTGAAAGCGCAACTCCT
  			GAGTCTGGCCCAGGTAGCGAAC
  			CTGCTACCTCCGGCTCTGAGAC
  			TCCAGGTACCTCTGAAAGCGCA
  			ACCCCGGAATCTGGTCCAGGTA
  			GCGAACCTGCAACCTCTGGCTC
  			TGAAACCCCAGGTACCTCTGAA
  			AGCGCTACTCCTGAATCTGGCC
  			CAGGTACTTCTACTGAACCGTC
  			CGAGGGCAGCGCACCAGGTACT
  			TCTGAAAGCGCTACTCCTGAGT
  			CCGGCCCAGGTAGCCCGGCTGG
  			CTCTCCGACTTCCACCGAGGAA
  			GGTAGCCCGGCTGGCTCTCCAA
  			CTTCTACTGAAGAAGGTAGCCC
  			GGCAGGCTCTCCGACCTCTACT
  			GAGGAAGGTACTTCTGAAAGCG
  			CAACCCCGGAGTCCGGCCCAGG
  			TACCTCTACCGAACCGTCTGAG
  			GGCAGCGCACCAGGTACCTCTG
  			AAAGCGCAACTCCTGAGTCTGG
  			CCCAGGTAGCGAACCTGCTACC
  			TCCGGCTCTGAGACTCCAGGTA
  			CCTCTGAAAGCGCAACCCCGGA
  			ATCTGGTCCAGGTAGCGAACCT
  			GCAACCTCTGGCTCTGAAACCC
  			CAGGTACCTCTGAAAGCGCTAC
  			TCCTGAATCTGGCCCAGGTACT
  			TCTACTGAACCGTCCGAGGGCA
  			GCGCACCAGGTAGCCCTGCTGG
  			CTCTCCAACCTCCACCGAAGAA
  			GGTACCTCTGAAAGCGCAACCC
  			CTGAATCCGGCCCAGGTAGCGA
  			ACCGGCAACCTCCGGTTCTGAA
  			ACCCCAGGTACTTCTGAAAGCG
  			CTACTCCTGAGTCCGGCCCAGG
  			TAGCCCGGCTGGCTCTCCGACT
  			TCCACCGAGGAAGGTAGCCCGG
  			CTGGCTCTCCAACTTCTACTGA
  			AGAAGGTACTTCTACCGAACCT
  			TCCGAGGGCAGCGCACCAGGTA
  			CTTCTGAAAGCGCTACCCCTGA
  			GTCCGGCCCAGGTACTTCTGAA
  			AGCGCTACTCCTGAATCCGGTC
  			CAGGTACTTCTGAAAGCGCTAC
  			CCCGGAATCTGGCCCAGGTAGC
  			GAACCGGCTACTTCTGGTTCTG
  			AAACCCCAGGTAGCGAACCGGC
  			TACCTCCGGTTCTGAAACTCCA
  			GGTAGCCCAGCAGGCTCTCCGA
  			CTTCCACTGAGGAAGGTACTTC
  			TACTGAACCTTCCGAAGGCAGC
  			GCACCAGGTACCTCTACTGAAC
  			CTTCTGAGGGCAGCGCTCCAGG
  			TAGCGAACCTGCAACCTCTGGC
  			TCTGAAACCCCAGGTACCTCTG
  			AAAGCGCTACTCCTGAATCTGG
  			CCCAGGTACTTCTACTGAACCG
  			TCCGAGGGCAGCGCACCA
  FVII-	ANAFLEELRPGSLE	734	GCCAACGCGTTCCTGGAGGAGC	735
  MMP-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  17-	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  AE288	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	APLGLRLRGGTSE		AGGTCATCATCCCCAGCACGTA
  	SATPESGPGSEPAT		CGTCCCGGGCACCACCAACCAC
  	SGSETPGTSESATP		GACATCGCGCTGCTCCGCCTGC
  	ESGPGSEPATSGSE		ACCAGCCCGTGGTCCTCACTGA
  	TPGTSESATPESGP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGS		CCCGAACGGACGTTCTCTGAGA
  	PAGSPTSTEEGTSE		GGACGCTGGCCTTCGTGCGCTT
  	SATPESGPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGTSESATP		CAGCTGCTGGACCGTGGCGCCA
  	ESGPGSPAGSPTST		CGGCCCTGGAGCTCATGGTCCT
  	EEGSPAGSPTSTEE		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGT		CAGGACTGCCTGCAGCAGTCAC
  	SESATPESGPGTSE		GGAAGGTGGGAGACTCCCCAA
  	SATPESGPGTSESA		ATATCACGGAGTACATGTTCTG
  	TPESGPGSEPATSG		TGCCGGCTACTCGGATGGCAGC
  	SETPGSEPATSGSE		AAGGACTCCTGCAAGGGGGAC
  	TPGSPAGSPTSTEE		AGTGGAGGCCCACATGCCACCC
  	GTSTEPSEGSAPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGSEP		GACGGGCATCGTCAGCTGGGGC
  	ATSGSETPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSTEPSE		ACTTTGGGGTGTACACCAGGGT
  	GSAP		CTCCCAGTACATCGAGTGGCTG
  			CAAAAGCTCATGCGCTCAGAGC
  			CACGCCCAGGAGTCCTCCTGCG
  			AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-	ANAFLEELRPGSLE	736	GCCAACGCGTTCCTGGAGGAGC	737
  MMP-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  17-	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  AE864	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	APLGLRLRGGSPA		AGGTCATCATCCCCAGCACGTA
  	GSPTSTEEGTSESA		CGTCCCGGGCACCACCAACCAC
  	TPESGPGTSTEPSE		GACATCGCGCTGCTCCGCCTGC
  	GSAPGSPAGSPTST		ACCAGCCCGTGGTCCTCACTGA
  	EEGTSTEPSEGSAP		CCATGTGGTGCCCCTCTGCCTG
  	GTSTEPSEGSAPGT		CCCGAACGGACGTTCTCTGAGA
  	SESATPESGPGSEP		GGACGCTGGCCTTCGTGCGCTT
  	ATSGSETPGSEPAT		CTCATTGGTCAGCGGCTGGGGC
  	SGSETPGSPAGSPT		CAGCTGCTGGACCGTGGCGCCA
  	STEEGTSESATPES		CGGCCCTGGAGCTCATGGTCCT
  	GPGTSTEPSEGSAP		CAACGTGCCCCGGCTGATGACC
  	GTSTEPSEGSAPGS		CAGGACTGCCTGCAGCAGTCAC
  	PAGSPTSTEEGTST		GGAAGGTGGGAGACTCCCCAA
  	EPSEGSAPGTSTEP		ATATCACGGAGTACATGTTCTG
  	SEGSAPGTSESATP		TGCCGGCTACTCGGATGGCAGC
  	ESGPGTSTEPSEGS		AAGGACTCCTGCAAGGGGGAC
  	APGTSESATPESGP		AGTGGAGGCCCACATGCCACCC
  	GSEPATSGSETPGT		ACTACCGGGGCACGTGGTACCT
  	STEPSEGSAPGTST		GACGGGCATCGTCAGCTGGGGC
  	EPSEGSAPGTSESA		CAGGGCTGCGCAACCGTGGGCC
  	TPESGPGTSESATP		ACTTTGGGGTGTACACCAGGGT
  	ESGPGSPAGSPTST		CTCCCAGTACATCGAGTGGCTG
  	EEGTSESATPESGP		CAAAAGCTCATGCGCTCAGAGC
  	GSEPATSGSETPGT		CACGCCCAGGAGTCCTCCTGCG
  	SESATPESGPGTST		AGCCCCATTTCCCGGT0GGTGGT
  	EPSEGSAPGTSTEP		AGCCCGGCTGGCTCTCCTACCT
  	SEGSAPGTSTEPSE		CTACTGAGGAAGGTACTTCTGA
  	GSAPGTSTEPSEGS		AAGCGCTACTCCTGAGTCTGGT
  	APGTSTEPSEGSAP		CCAGGTACCTCTACTGAACCGT
  	GTSTEPSEGSAPGS		CCGAAGGTAGCGCTCCAGGTAG
  	PAGSPTSTEEGTST		CCCAGCAGGCTCTCCGACTTCC
  	EPSEGSAPGTSESA		ACTGAGGAAGGTACTTCTACTG
  	TPESGPGSEPATSG		AACCTTCCGAAGGCAGCGCACC
  	SETPGTSESATPES		AGGTACCTCTACTGAACCTTCT
  	GPGSEPATSGSETP		GAGGGCAGCGCTCCAGGTACTT
  	GTSESATPESGPGT		CTGAAAGCGCTACCCCGGAATC
  	STEPSEGSAPGTSE		TGGCCCAGGTAGCGAACCGGCT
  	SATPESGPGSPAGS		ACTTCTGGTTCTGAAACCCCAG
  	PTSTEEGSPAGSPT		GTAGCGAACCGGCTACCTCCGG
  	STEEGSPAGSPTST		TTCTGAAACTCCAGGTAGCCCG
  	EEGTSESATPESGP		GCAGGCTCTCCGACCTCTACTG
  	GTSTEPSEGSAPGT		AGGAAGGTACTTCTGAAAGCGC
  	SESATPESGPGSEP		AACCCCGGAGTCCGGCCCAGGT
  	ATSGSETPGTSESA		ACCTCTACCGAACCGTCTGAGG
  	TPESGPGSEPATSG		GCAGCGCACCAGGTACTTCTAC
  	SETPGTSESATPES		CGAACCGTCCGAGGGTAGCGCA
  	GPGTSTEPSEGSAP		CCAGGTAGCCCAGCAGGTTCTC
  	GSPAGSPTSTEEGT		CTACCTCCACCGAGGAAGGTAC
  	SESATPESGPGSEP		TTCTACCGAACCGTCCGAGGGT
  	ATSGSETPGTSESA		AGCGCACCAGGTACCTCTACTG
  	TPESGPGSPAGSPT		AACCTTCTGAGGGCAGCGCTCC
  	STEEGSPAGSPTST		AGGTACTTCTGAAAGCGCTACC
  	EEGTSTEPSEGSAP		CCGGAGTCCGGTCCAGGTACTT
  	GTSESATPESGPGT		CTACTGAACCGTCCGAAGGTAG
  	SESATPESGPGTSE		CGCACCAGGTACTTCTGAAAGC
  	SATPESGPGSEPAT		GCAACCCCTGAATCCGGTCCAG
  	SGSETPGSEPATSG		GTAGCGAACCGGCTACTTCTGG
  	SETPGSPAGSPTST		CTCTGAGACTCCAGGTACTTCT
  	EEGTSTEPSEGSAP		ACCGAACCGTCCGAAGGTAGCG
  	GTSTEPSEGSAPGS		CACCAGGTACTTCTACTGAACC
  	EPATSGSETPGTSE		GTCTGAAGGTAGCGCACCAGGT
  	SATPESGPGTSTEP		ACTTCTGAAAGCGCAACCCCGG
  	SEGSAP		AATCCGGCCCAGGTACCTCTGA
  			AAGCGCAACCCCGGAGTCCGGC
  			CCAGGTAGCCCTGCTGGCTCTC
  			CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  FIX-	YNSGKLEEFVQGN	738	TATAATTCAGGTAAATTGGAAG	739
  MMP-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  17-FXIa	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGA		AATTACAGTTGTCGCAGGTGAA
  	PLGLRLRGKLTRA		CATAATATTGAGGAGACAGAAC
  	ET		ATACAGAGCAAAAGCGAAATG
  			TGATTCGAATTATTCCTCACCAC
  			AACTACAATGCAGCTATTAATA
  			AGTACAACCATGACATTGCCCT
  			TCTGGAACTGGACGAACCCTTA
  			GTGCTAAACAGCTACGTTACAC
  			CTATTTGCATTGCTGACAAGGA
  			ATACACGAACATCTTCCTCAAA
  			TTTGGATCTGGCTATGTAAGTG
  			GCTGGGGAAGAGTCTTCCACAA
  			AGGGAGATCAGCTTTAGTTCTT
  			CAGTACCTTAGAGTTCCACTTG
  			TTGACCGAGCCACATGTCTTCG
  			ATCTACAAAGTTCACCATCTAT
  			AACAACATGTTCTGTGCTGGCT
  			TCCATGAAGGAGGTAGAGATTC
  			ATGTCAAGGAGATAGTGGGGG
  			ACCCCATGTTACTGAAGTGGAA
  			GGGACCAGTTTCTTAACTGGAA
  			TTATTAGCTGGGGTGAAGAGTG
  			TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0GGT0
  FIX-	YNSGKLEEFVQGN	740	TATAATTCAGGTAAATTGGAAG	741
  MMP-	LERECMEEKCSFE		AGTTTGTTCAAGGGAACCTTGA
  17-	EAREVFENTERTT		GAGAGAATGTATGGAAGAAAA
  Elastase	EFWKQYVDGDQC		GTGTAGTTTTGAAGAAGCACGA
  	ESNPCLNGGSCKD		GAAGTTTTTGAAAACACTGAAA
  	DINSYECWCPFGF		GAACAACTGAATTTTGGAAGCA
  	EGKNCELDVTCNI		GTATGTTGATGGAGATCAGTGT
  	KNGRCEQFCKNSA		GAGTCCAATCCATGTTTAAATG
  	DNKVVCSCTEGYR		GCGGCAGTTGCAAGGATGACAT
  	LAENQKSCEPAVP		TAATTCCTATGAATGTTGGTGTC
  	FPCGRVSVSQTSK		CCTTTGGATTTGAAGGAAAGAA
  	LTRAETVFPDVDY		CTGTGAATTAGATGTAACATGT
  	VNSTEAETILDNIT		AACATTAAGAATGGCAGATGCG
  	QSTQSFNDFTRVV		AGCAGTTTTGTAAAAATAGTGC
  	GGEDAKPGQFPW		TGATAACAAGGTGGTTTGCTCC
  	QVVLNGKVDAFC		TGTACTGAGGGATATCGACTTG
  	GGSIVNEKWIVTA		CAGAAAACCAGAAGTCCTGTGA
  	AHCVETGVKITVV		ACCAGCAGTGCCATTTCCATGT
  	AGEHNIEETEHTE		GGAAGAGTTTCTGTTTCACAAA
  	QKRNVIRIIPHHNY		CTTCTAAGCTCACCCGTGCTGA
  	NAAINKYNHDIAL		GACTGTTTTTCCTGATGTGGACT
  	LELDEPLVLNSYV		ATGTAAATTCTACTGAAGCTGA
  	TPICIADKEYTNIFL		AACCATTTTGGATAACATCACT
  	KFGSGYVSGWGR		CAAAGCACCCAATCATTTAATG
  	VFHKGRSALVLQY		ACTTCACTCGGGTTGTTGGTGG
  	LRVPLVDRATCLR		AGAAGATGCCAAACCAGGTCA
  	STKFTIYNNMFCA		ATTCCCTTGGCAGGTTGTTTTGA
  	GFHEGGRDSCQGD		ATGGTAAAGTTGATGCATTCTG
  	SGGPHVTEVEGTS		TGGAGGCTCTATCGTTAATGAA
  	FLTGIISWGEECAM		AAATGGATTGTAACTGCTGCCC
  	KGKYGIYTKVSRY		ACTGTGTTGAAACTGGTGTTAA
  	VNWIKEKTKLTGA		AATTACAGTTGTCGCAGGTGAA
  	PLGLRLR		CATAATATTGAGGAGACAGAAC
  			ATACAGAGCAAAAGCGAAATG
  			TGATTCGAATTATTCCTCACCAC
  			AACTACAATGCAGCTATTAATA
  			AGTACAACCATGACATTGCCCT
  			TCTGGAACTGGACGAACCCTTA
  			GTGCTAAACAGCTACGTTACAC
  			CTATTTGCATTGCTGACAAGGA
  			ATACACGAACATCTTCCTCAAA
  			TTTGGATCTGGCTATGTAAGTG
  			GCTGGGGAAGAGTCTTCCACAA
  			AGGGAGATCAGCTTTAGTTCTT
  			CAGTACCTTAGAGTTCCACTTG
  			TTGACCGAGCCACATGTCTTCG
  			ATCTACAAAGTTCACCATCTAT
  			AACAACATGTTCTGTGCTGGCT
  			TCCATGAAGGAGGTAGAGATTC
  			ATGTCAAGGAGATAGTGGGGG
  			ACCCCATGTTACTGAAGTGGAA
  			GGGACCAGTTTCTTAACTGGAA
  			TTATTAGCTGGGGTGAAGAGTG
  			TGCAATGAAAGGCAAATATGGA
  			ATATATACCAAGGTATCCCGGT
  			ATGTCAACTGGATTAAGGAAAA
  			AACAAAGCTCACTGGGGT0
  FVII-FIX	ANAFLEELRPGSLE	742	GCCAACGCGTTCCTGGAGGAGC	743
  AP-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE288	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	KLTRAETVFPDVD		AGGTCATCATCCCCAGCACGTA
  	YVNSTEAETILDNI		CGTCCCGGGCACCACCAACCAC
  	TQSTQSFNDFTRV		GACATCGCGCTGCTCCGCCTGC
  	VGGE		ACCAGCCCGTGGTCCTCACTGA
  	GGTSESATPESGPG		CCATGTGGTGCCCCTCTGCCTG
  	SEPATSGSETPGTS		CCCGAACGGACGTTCTCTGAGA
  	ESATPESGPGSEPA		GGACGCTGGCCTTCGTGCGCTT
  	TSGSETPGTSESAT		CTCATTGGTCAGCGGCTGGGGC
  	PESGPGTSTEPSEG		CAGCTGCTGGACCGTGGCGCCA
  	SAPGSPAGSPTSTE		CGGCCCTGGAGCTCATGGTCCT
  	EGTSESATPESGPG		CAACGTGCCCCGGCTGATGACC
  	SEPATSGSETPGTS		CAGGACTGCCTGCAGCAGTCAC
  	ESATPESGPGSPAG		GGAAGGTGGGAGACTCCCCAA
  	SPTSTEEGSPAGSP		ATATCACGGAGTACATGTTCTG
  	TSTEEGTSTEPSEG		TGCCGGCTACTCGGATGGCAGC
  	SAPGTSESATPESG		AAGGACTCCTGCAAGGGGGAC
  	PGTSESATPESGPG		AGTGGAGGCCCACATGCCACCC
  	TSESATPESGPGSE		ACTACCGGGGCACGTGGTACCT
  	PATSGSETPGSEPA		GACGGGCATCGTCAGCTGGGGC
  	TSGSETPGSPAGSP		CAGGGCTGCGCAACCGTGGGCC
  	TSTEEGTSTEPSEG		ACTTTGGGGTGTACACCAGGGT
  	SAPGTSTEPSEGSA		CTCCCAGTACATCGAGTGGCTG
  	PGSEPATSGSETPG		CAAAAGCTCATGCGCTCAGAGC
  	TSESATPESGPGTS		CACGCCCAGGAGTCCTCCTGCG
  	TEPSEGSAP		AGCCCCATTTCCCGGT0GGTGGT
  			ACCTCTGAAAGCGCAACTCCTG
  			AGTCTGGCCCAGGTAGCGAACC
  			TGCTACCTCCGGCTCTGAGACT
  			CCAGGTACCTCTGAAAGCGCAA
  			CCCCGGAATCTGGTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCAGGTAGC
  			CCTGCTGGCTCTCCAACCTCCA
  			CCGAAGAAGGTACCTCTGAAAG
  			CGCAACCCCTGAATCCGGCCCA
  			GGTAGCGAACCGGCAACCTCCG
  			GTTCTGAAACCCCAGGTACTTC
  			TGAAAGCGCTACTCCTGAGTCC
  			GGCCCAGGTAGCCCGGCTGGCT
  			CTCCGACTTCCACCGAGGAAGG
  			TAGCCCGGCTGGCTCTCCAACT
  			TCTACTGAAGAAGGTACTTCTA
  			CCGAACCTTCCGAGGGCAGCGC
  			ACCAGGTACTTCTGAAAGCGCT
  			ACCCCTGAGTCCGGCCCAGGTA
  			CTTCTGAAAGCGCTACTCCTGA
  			ATCCGGTCCAGGTACTTCTGAA
  			AGCGCTACCCCGGAATCTGGCC
  			CAGGTAGCGAACCGGCTACTTC
  			TGGTTCTGAAACCCCAGGTAGC
  			GAACCGGCTACCTCCGGTTCTG
  			AAACTCCAGGTAGCCCAGCAGG
  			CTCTCCGACTTCCACTGAGGAA
  			GGTACTTCTACTGAACCTTCCG
  			AAGGCAGCGCACCAGGTACCTC
  			TACTGAACCTTCTGAGGGCAGC
  			GCTCCAGGTAGCGAACCTGCAA
  			CCTCTGGCTCTGAAACCCCAGG
  			TACCTCTGAAAGCGCTACTCCT
  			GAATCTGGCCCAGGTACTTCTA
  			CTGAACCGTCCGAGGGCAGCGC
  			ACCA
  FVII-FIX	ANAFLEELRPGSLE	744	GCCAACGCGTTCCTGGAGGAGC	745
  AP-	RECKEEQCSFEEA		TACGGCCGGGCTCCCTGGAGAG
  AE864	REIFKDAERTKLF		GGAGTGCAAGGAGGAGCAGTG
  	WISYSDGDQCASS		CTCCTTCGAGGAGGCCCGGGAG
  	PCQNGGSCKDQLQ		ATCTTCAAGGACGCGGAGAGGA
  	SYICFCLPAFEGRN		CGAAGCTGTTCTGGATTTCTTAC
  	CETHKDDQLICVN		AGTGATGGGGACCAGTGTGCCT
  	ENGGCEQYCSDHT		CAAGTCCATGCCAGAATGGGGG
  	GTKRSCRCHEGYS		CTCCTGCAAGGACCAGCTCCAG
  	LLADGVSCTPTVE		TCCTATATCTGCTTCTGCCTCCC
  	YPCGKIPILEKRNA		TGCCTTCGAGGGCCGGAACTGT
  	SKPQGRIVGGKVC		GAGACGCACAAGGATGACCAG
  	PKGECPWQVLLLV		CTGATCTGTGTGAACGAGAACG
  	NGAQLCGGTLINTI		GCGGCTGTGAGCAGTACTGCAG
  	WVVSAAHCFDKIK		TGACCACACGGGCACCAAGCGC
  	NWRNLIAVLGEHD		TCCTGTCGGTGCCACGAGGGGT
  	LSEHDGDEQSRRV		ACTCTCTGCTGGCAGACGGGGT
  	AQVIIPSTYVPGTT		GTCCTGCACACCCACAGTTGAA
  	NHDIALLRLHQPV		TATCCATGTGGAAAAATACCTA
  	VLTDHVVPLCLPE		TTCTAGAAAAAAGAAATGCCAG
  	RTFSERTLAFVRFS		CAAACCCCAAGGCCGAATTGTG
  	LVSGWGQLLDRG		GGGGGCAAGGTGTGCCCCAAA
  	ATALELMVLNVPR		GGGGAGTGTCCATGGCAGGTCC
  	LMTQDCLQQSRK		TGTTGTTGGTGAATGGAGCTCA
  	VGDSPNITEYMFC		GTTGTGTGGGGGGACCCTGATC
  	AGYSDGSKDSCKG		AACACCATCTGGGTGGTCTCCG
  	DSGGPHATHYRGT		CGGCCCACTGTTTCGACAAAAT
  	WYLTGIVSWGQG		CAAGAACTGGAGGAACCTGATC
  	CATVGHFGVYTRV		GCGGTGCTGGGCGAGCACGACC
  	SQYIEWLQKLMRS		TCAGCGAGCACGACGGGGATG
  	EPRPGVLLRAPFPG		AGCAGAGCCGGCGGGTGGCGC
  	KLTRAETVFPDVD		AGGTCATCATCCCCAGCACGTA
  	YVNSTEAETILDNI		CGTCCCGGGCACCACCAACCAC
  	TQSTQSFNDFTRV		GACATCGCGCTGCTCCGCCTGC
  	VGGE		ACCAGCCCGTGGTCCTCACTGA
  	GGSPAGSPTSTEEG		CCATGTGGTGCCCCTCTGCCTG
  	TSESATPESGPGTS		CCCGAACGGACGTTCTCTGAGA
  	TEPSEGSAPGSPAG		GGACGCTGGCCTTCGTGCGCTT
  	SPTSTEEGTSTEPS		CTCATTGGTCAGCGGCTGGGGC
  	EGSAPGTSTEPSEG		CAGCTGCTGGACCGTGGCGCCA
  	SAPGTSESATPESG		CGGCCCTGGAGCTCATGGTCCT
  	PGSEPATSGSETPG		CAACGTGCCCCGGCTGATGACC
  	SEPATSGSETPGSP		CAGGACTGCCTGCAGCAGTCAC
  	AGSPTSTEEGTSES		GGAAGGTGGGAGACTCCCCAA
  	ATPESGPGTSTEPS		ATATCACGGAGTACATGTTCTG
  	EGSAPGTSTEPSEG		TGCCGGCTACTCGGATGGCAGC
  	SAPGSPAGSPTSTE		AAGGACTCCTGCAAGGGGGAC
  	EGTSTEPSEGSAPG		AGTGGAGGCCCACATGCCACCC
  	TSTEPSEGSAPGTS		ACTACCGGGGCACGTGGTACCT
  	ESATPESGPGTSTE		GACGGGCATCGTCAGCTGGGGC
  	PSEGSAPGTSESAT		CAGGGCTGCGCAACCGTGGGCC
  	PESGPGSEPATSGS		ACTTTGGGGTGTACACCAGGGT
  	ETPGTSTEPSEGSA		CTCCCAGTACATCGAGTGGCTG
  	PGTSTEPSEGSAPG		CAAAAGCTCATGCGCTCAGAGC
  	TSESATPESGPGTS		CACGCCCAGGAGTCCTCCTGCG
  	ESATPESGPGSPAG		AGCCCCATTTCCCGGT0GGTGGT
  	SPTSTEEGTSESAT		AGCCCGGCTGGCTCTCCTACCT
  	PESGPGSEPATSGS		CTACTGAGGAAGGTACTTCTGA
  	ETPGTSESATPESG		AAGCGCTACTCCTGAGTCTGGT
  	PGTSTEPSEGSAPG		CCAGGTACCTCTACTGAACCGT
  	TSTEPSEGSAPGTS		CCGAAGGTAGCGCTCCAGGTAG
  	TEPSEGSAPGTSTE		CCCAGCAGGCTCTCCGACTTCC
  	PSEGSAPGTSTEPS		ACTGAGGAAGGTACTTCTACTG
  	EGSAPGTSTEPSEG		AACCTTCCGAAGGCAGCGCACC
  	SAPGSPAGSPTSTE		AGGTACCTCTACTGAACCTTCT
  	EGTSTEPSEGSAPG		GAGGGCAGCGCTCCAGGTACTT
  	TSESATPESGPGSE		CTGAAAGCGCTACCCCGGAATC
  	PATSGSETPGTSES		TGGCCCAGGTAGCGAACCGGCT
  	ATPESGPGSEPATS		ACTTCTGGTTCTGAAACCCCAG
  	GSETPGTSESATPE		GTAGCGAACCGGCTACCTCCGG
  	SGPGTSTEPSEGSA		TTCTGAAACTCCAGGTAGCCCG
  	PGTSESATPESGPG		GCAGGCTCTCCGACCTCTACTG
  	SPAGSPTSTEEGSP		AGGAAGGTACTTCTGAAAGCGC
  	AGSPTSTEEGSPAG		AACCCCGGAGTCCGGCCCAGGT
  	SPTSTEEGTSESAT		ACCTCTACCGAACCGTCTGAGG
  	PESGPGTSTEPSEG		GCAGCGCACCAGGTACTTCTAC
  	SAPGTSESATPESG		CGAACCGTCCGAGGGTAGCGCA
  	PGSEPATSGSETPG		CCAGGTAGCCCAGCAGGTTCTC
  	TSESATPESGPGSE		CTACCTCCACCGAGGAAGGTAC
  	PATSGSETPGTSES		TTCTACCGAACCGTCCGAGGGT
  	ATPESGPGTSTEPS		AGCGCACCAGGTACCTCTACTG
  	EGSAPGSPAGSPTS		AACCTTCTGAGGGCAGCGCTCC
  	TEEGTSESATPESG		AGGTACTTCTGAAAGCGCTACC
  	PGSEPATSGSETPG		CCGGAGTCCGGTCCAGGTACTT
  	TSESATPESGPGSP		CTACTGAACCGTCCGAAGGTAG
  	AGSPTSTEEGSPAG		CGCACCAGGTACTTCTGAAAGC
  	SPTSTEEGTSTEPS		GCAACCCCTGAATCCGGTCCAG
  	EGSAPGTSESATPE		GTAGCGAACCGGCTACTTCTGG
  	SGPGTSESATPESG		CTCTGAGACTCCAGGTACTTCT
  	PGTSESATPESGPG		ACCGAACCGTCCGAAGGTAGCG
  	SEPATSGSETPGSE		CACCAGGTACTTCTACTGAACC
  	PATSGSETPGSPAG		GTCTGAAGGTAGCGCACCAGGT
  	SPTSTEEGTSTEPS		ACTTCTGAAAGCGCAACCCCGG
  	EGSAPGTSTEPSEG		AATCCGGCCCAGGTACCTCTGA
  	SAPGSEPATSGSET		AAGCGCAACCCCGGAGTCCGGC
  	PGTSESATPESGPG		CCAGGTAGCCCTGCTGGCTCTC
  	TSTEPSEGSAP		CAACCTCCACCGAAGAAGGTAC
  			CTCTGAAAGCGCAACCCCTGAA
  			TCCGGCCCAGGTAGCGAACCGG
  			CAACCTCCGGTTCTGAAACCCC
  			AGGTACCTCTGAAAGCGCTACT
  			CCGGAGTCTGGCCCAGGTACCT
  			CTACTGAACCGTCTGAGGGTAG
  			CGCTCCAGGTACTTCTACTGAA
  			CCGTCCGAAGGTAGCGCACCAG
  			GTACTTCTACCGAACCGTCCGA
  			AGGCAGCGCTCCAGGTACCTCT
  			ACTGAACCTTCCGAGGGCAGCG
  			CTCCAGGTACCTCTACCGAACC
  			TTCTGAAGGTAGCGCACCAGGT
  			ACTTCTACCGAACCGTCCGAGG
  			GTAGCGCACCAGGTAGCCCAGC
  			AGGTTCTCCTACCTCCACCGAG
  			GAAGGTACTTCTACCGAACCGT
  			CCGAGGGTAGCGCACCAGGTAC
  			CTCTGAAAGCGCAACTCCTGAG
  			TCTGGCCCAGGTAGCGAACCTG
  			CTACCTCCGGCTCTGAGACTCC
  			AGGTACCTCTGAAAGCGCAACC
  			CCGGAATCTGGTCCAGGTAGCG
  			AACCTGCAACCTCTGGCTCTGA
  			AACCCCAGGTACCTCTGAAAGC
  			GCTACTCCTGAATCTGGCCCAG
  			GTACTTCTACTGAACCGTCCGA
  			GGGCAGCGCACCAGGTACTTCT
  			GAAAGCGCTACTCCTGAGTCCG
  			GCCCAGGTAGCCCGGCTGGCTC
  			TCCGACTTCCACCGAGGAAGGT
  			AGCCCGGCTGGCTCTCCAACTT
  			CTACTGAAGAAGGTAGCCCGGC
  			AGGCTCTCCGACCTCTACTGAG
  			GAAGGTACTTCTGAAAGCGCAA
  			CCCCGGAGTCCGGCCCAGGTAC
  			CTCTACCGAACCGTCTGAGGGC
  			AGCGCACCAGGTACCTCTGAAA
  			GCGCAACTCCTGAGTCTGGCCC
  			AGGTAGCGAACCTGCTACCTCC
  			GGCTCTGAGACTCCAGGTACCT
  			CTGAAAGCGCAACCCCGGAATC
  			TGGTCCAGGTAGCGAACCTGCA
  			ACCTCTGGCTCTGAAACCCCAG
  			GTACCTCTGAAAGCGCTACTCC
  			TGAATCTGGCCCAGGTACTTCT
  			ACTGAACCGTCCGAGGGCAGCG
  			CACCAGGTAGCCCTGCTGGCTC
  			TCCAACCTCCACCGAAGAAGGT
  			ACCTCTGAAAGCGCAACCCCTG
  			AATCCGGCCCAGGTAGCGAACC
  			GGCAACCTCCGGTTCTGAAACC
  			CCAGGTACTTCTGAAAGCGCTA
  			CTCCTGAGTCCGGCCCAGGTAG
  			CCCGGCTGGCTCTCCGACTTCC
  			ACCGAGGAAGGTAGCCCGGCTG
  			GCTCTCCAACTTCTACTGAAGA
  			AGGTACTTCTACCGAACCTTCC
  			GAGGGCAGCGCACCAGGTACTT
  			CTGAAAGCGCTACCCCTGAGTC
  			CGGCCCAGGTACTTCTGAAAGC
  			GCTACTCCTGAATCCGGTCCAG
  			GTACTTCTGAAAGCGCTACCCC
  			GGAATCTGGCCCAGGTAGCGAA
  			CCGGCTACTTCTGGTTCTGAAA
  			CCCCAGGTAGCGAACCGGCTAC
  			CTCCGGTTCTGAAACTCCAGGT
  			AGCCCAGCAGGCTCTCCGACTT
  			CCACTGAGGAAGGTACTTCTAC
  			TGAACCTTCCGAAGGCAGCGCA
  			CCAGGTACCTCTACTGAACCTT
  			CTGAGGGCAGCGCTCCAGGTAG
  			CGAACCTGCAACCTCTGGCTCT
  			GAAACCCCAGGTACCTCTGAAA
  			GCGCTACTCCTGAATCTGGCCC
  			AGGTACTTCTACTGAACCGTCC
  			GAGGGCAGCGCACCA
  *Sequence name reflects N- to C-terminus configuration of the CF, cleavage sequence and XTEN components

• TABLE 43
  Exemplary FVII variants incorporating FIX AP sequence,
  cleavage sequences and XTEN**

  FVII		SEQ
  Construct		ID
  Name*	Amino Acid Sequence	NO:
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	746
  EGF2)-(AP)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGS
  	KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQ
  	GRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQ
  	LLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYS
  	DGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  	VSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	747
  EGF2)-(AP)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  AE288	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGS
  	KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQ
  	GRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQ
  	LLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYS
  	DGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  	VSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPESGPGSEPATSGS
  	ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  	GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP
  	AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA
  	TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS
  	TEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP
  	GTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	748
  EGF2)-(AP)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  AE864	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNGS
  	KLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEGSKPQ
  	GRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQ
  	LLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYS
  	DGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR
  	VSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTSTEEGTSESATPES
  	GPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG
  	TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE
  	SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS
  	EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES
  	GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
  	TSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE
  	SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS
  	EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
  	APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG
  	TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
  	GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT
  	PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
  	TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
  	SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
  	GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSE
  	TPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	749
  EGF2)-(K142-V149)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	KLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  	FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSP
  	NITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGC
  	ATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	750
  EGF2)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  (K142-V149)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  FVII(Pro)-	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  AE288	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	KLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  	FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSP
  	NITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGC
  	ATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESATPE
  	SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
  	GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS
  	ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESA
  	TPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS
  	ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP
  	GTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	751
  EGF2)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  (K142-V149)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  FVII(Pro)-	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  AE864	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	KLTRAETVGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLA
  	FVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSP
  	NITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGC
  	ATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSPTS
  	TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
  	GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
  	AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS
  	PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
  	SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP
  	GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE
  	PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP
  	SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS
  	TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
  	GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP
  	AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
  	SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS
  	ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP
  	GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS
  	TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT
  	SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG
  	SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	752
  EGF2)-(D177-G184)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	DFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTL
  	AFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
  	SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQ
  	GCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	753
  EGF2)-(D177-G184)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  AE288	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	DFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTL
  	AFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
  	SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQ
  	GCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGTSESAT
  	PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG
  	TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE
  	SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS
  	GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSE
  	TPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	754
  EGF2)-(D177-G184)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  AE864	SKPQIVGGKVCPKGECPWQVLLLVNGAQLCGGGRTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNG
  	DFTRVVGGGSKPQGRHDIALLRLHQPVVLTDHVVPLCLPERTFSERTL
  	AFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
  	SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQ
  	GCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGGSPAGSP
  	TSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG
  	SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS
  	EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS
  	APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
  	SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
  	TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
  	SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTST
  	EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS
  	GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
  	GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
  	TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP
  	ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
  	EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	755
  EGF2)-(D177-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  	SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  	EWLQKLMRSEPRPGVLLRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	756
  EGF2)-(D177-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  KLTRAET-	SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  AE288	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  (‘KLTRAET’	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  disclosed as	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  SEQ ID NO:	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  6)	EWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPATSGSETPGTSE
  	SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
  	TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
  	EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
  	TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
  	EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
  	EGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	757
  EGF2)-(D177-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  KLTRAET-	SKDFTRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  AE864(‘KLTRAET’	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  disclosed as	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  SEQ ID NO:	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  6)	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  	EWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTE
  	EGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
  	STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
  	SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
  	STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA
  	PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
  	SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA
  	TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
  	EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG
  	SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
  	PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE
  	PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG
  	SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
  	PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	758
  EGF2)-(K142-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL
  	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGEC
  	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	759
  EGF2)-(K142-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL
  KLTRAET-	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGEC
  AE288(‘KLTR	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  AET’	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  disclosed as	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  SEQ ID NO:	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  6)	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
  	TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
  	PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	760
  EGF2)-(K142-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRKL
  KLTRAET-	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGEC
  AE864(‘KLTR	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  AET’	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  disclosed as	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  SEQ ID NO:	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  6)	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPS
  	EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
  	GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
  	TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
  	EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS
  	GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
  	TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
  	PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
  	SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
  	ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
  	EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
  	TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT
  	PESGPGTSTEPSEGSAPGSSS
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	761
  EGF2)-(R134-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGE
  	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	762
  EGF2)-(R134-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  KLTRAET-	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGE
  AE288(‘KLTR	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  AET’	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  disclosed as	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  SEQ ID NO:	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  6)	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE
  	EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS
  	PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
  	ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	763
  EGF2)-(R134-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  KLTRAET-	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIVGGKVCPKGE
  AE864(‘KLTR	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  AET’	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  disclosed as	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  SEQ ID NO:	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  6)	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTE
  	PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG
  	PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
  	STEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
  	TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
  	ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG
  	PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
  	ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
  	EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE
  	PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG
  	PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
  	ATPESGPGTSTEPSEGSAPGSSS
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	764
  EGF2)-(D177-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  	SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  	EWLQKLMRSEPRPGVLLRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	765
  EGF2)-(D177-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  KLTRAET-	SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  AE288(‘KLTR	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  AET’	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  disclosed as	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  SEQ ID NO:	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  6)	EWLQKLMRSEPRPGVLLRAPFPGTSESATPESGPGSEPATSGSETPGTSE
  	SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
  	TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
  	EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
  	TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST
  	EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
  	EGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	766
  EGF2)-(D177-T179)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNA
  KLTRAET-	SKDFTRVVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAH
  AE864(‘KLTR	CFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNH
  AET’	DIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDR
  disclosed as	GATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSK
  SEQ ID NO:	DSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYI
  6)	EWLQKLMRSEPRPGVLLRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTE
  	EGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
  	STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG
  	SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
  	STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA
  	PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT
  	SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA
  	TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE
  	GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE
  	EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG
  	SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
  	GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET
  	PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE
  	PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG
  	SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
  	PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSSS
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	767
  EGF2)-(K142-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL
  	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGEC
  	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	768
  EGF2)-(K142-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL
  KLTRAET-	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGEC
  AE288(‘KLTR	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  AET’	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  disclosed as	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  SEQ ID NO:	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  6)	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG
  	TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
  	PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
  	APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	769
  EGF2)-(K142-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGKIPILEKRKL
  KLTRAET-	TRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGEC
  AE864(‘KLTR	PWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEH
  AET’	DLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVP
  disclosed as	LCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQ
  SEQ ID NO:	DCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
  6)	WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
  	APFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPS
  	EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES
  	GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
  	TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
  	EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS
  	GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
  	GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
  	TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST
  	EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT
  	PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
  	SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP
  	ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
  	EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES
  	GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG
  	TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA
  	GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT
  	PESGPGTSTEPSEGSAPGSSS
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	770
  EGF2)-(R134-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGE
  	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	771
  EGF2)-(R134-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  KLTRAET-	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGE
  AE288(‘KLTR	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  AET’	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  disclosed as	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  SEQ ID NO:	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  6)	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFPGPEGPSKLTRAETGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE
  	EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS
  	PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES
  	ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE
  	GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
  FVII(Gla-	ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQ	772
  EGF2)-(R134-V181)-	CASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGG
  FVII(Pro)-	CEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVPFPCGRVSVSQTSK
  KLTRAET-	LTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGKVCPKGE
  AE864(‘KLTR	CPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGE
  AET’	HDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHV
  disclosed as	VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
  SEQ ID NO:	TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYR
  6)	GTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
  	LRAPFPGPEGPSKLTRAETGSPGSPAGSPTSTEEGTSESATPESGPGTSTE
  	PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP
  	ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG
  	PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
  	STEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
  	TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP
  	ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG
  	PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT
  	STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
  	ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE
  	EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
  	EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE
  	PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
  	ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG
  	PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS
  	PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES
  	ATPESGPGTSTEPSEGSAPGSSS
  *Sequence name reflects N- to C-terminus configuration of the FVII variant (Gla-EGF2 domains), FIX AP cleavage sequence, FVII protease domain, XTEN cleavage sequence and XTEN components (the latter when included)
  **Not all sequences incorporate XTEN

Claims (18)

1. An isolated factor VII polypeptide comprising an extended recombinant polypeptide (XTEN), said XTEN comprising at least 200 amino acid residues, wherein said factor IX polypeptide exhibits a terminal half-life that is longer than about 12 hours when administered to a subject.

2. The isolated factor IX polypeptide ofclaim 1, wherein the factor IX polypeptide exhibits at least 90% sequence identity compared to a sequence selected from Table 2 when optimally aligned.

3. The isolated factor IX polypeptide ofclaim 1 or2, wherein said factor VII is linked at its C-terminus to the XTEN.

4. The isolated factor IX polypeptide ofclaim 1 that is linked to the XTEN via a cleavage sequence that is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20.

5. The isolated factor IX polypeptide ofclaim 4, wherein cleavage at the cleavage sequence by the mammalian protease releases said XTEN from said factor IX polypeptide.

6. The isolated factor IX polypeptide ofclaim 1, wherein said XTEN is characterized in that:

(a) the cumulative total of XTEN amino acid residues is greater than 200 to about 3000 amino acid residues:

(b) the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN;

(c) the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN;

(d) the XTEN sequence has a subsequence score less than 10;

(e) the XTEN sequence has greater than 90% random coil formation as determined by GOR algorithm; and

(f) the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm.

7. The isolated factor IX polypeptide ofclaim 1 exhibiting an apparent molecular weight factor of at least about 4.

8. The isolated factor IX polypeptide ofclaim 1, wherein said XTEN exhibits at least 90% sequence identity to a comparable length of an amino acid sequence selected from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13.

9. The isolated factor IX polypeptide ofclaim 1 that is configured according to formula VII:

(Gla)-(XTEN)_u-(EGF1)-(XTEN)_v-(EGF2)-(AP1)-(XTEN)_w-(AP2)-(XTEN)_x-(Pro)-(S)_y-(XTEN)_z  VII

wherein independently for each occurrence,

(a) Gla is a Gla domain of factor IX;

(b) EGF1 is an EGF1 domain of factor IX;

(c) EGF2 is an EFG2 domain of factor IXI;

(d) AP1 is a portion of an activator peptide domain of factor IX;

(e) AP2 is a portion of an activator peptide domain of factor IX that includes at least a first cleavage sequence;

(f) PRO is a protease domain of factor IX;

(g) S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence;

(h) XTEN is an extended recombinant polypeptide that exhibits at least 90% sequence identity to a comparable length of an amino acid sequence selected from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13,

(i) u is either 0 or 1;

(j) v is either 0 or 1;

(k) x is either 0 or 1;

(l) y is either 0 or 1; and

(m) z is either 0 or 1, with the proviso that u+v+x+y+z≧1.

10. The isolated factor IX polypeptide ofclaim 9, wherein y=1 and S comprises a cleavage sequence that is cleavable by a mammalian protease selected from the group consisting of FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20.

11. The isolated factor IX polypeptide ofclaim 1 comprising more than one XTEN.

12. The isolated factor IX polypeptide ofclaim 1, wherein said XTEN is incorporated between any two adjacent domains contained in said factor IX, wherein said two adjacent domains are selected from the group consisting of Gla, EGF1, EGF2, AP, and peptidase S1 (Pro).

13. The isolated factor IX polypeptide ofclaim 1, characterized in that:

(i) it has a longer terminal half-life when administered to a mammal compared to the corresponding factor IX that lacks the XTEN when administered to a mammal at a comparable molar dose;

(ii) when a smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose regimen, the factor IX polypeptide achieves a comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN;

(iii) when a smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose regimen, the factor IX polypeptide achieves a comparable therapeutic effect as the corresponding factor IX that lacks the XTEN;

(iv) when the factor IX polypeptide is administered to a mammal less frequently in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal using an otherwise equivalent molar amount, the factor IX polypeptide achieves a comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN;

(v) when the factor IX polypeptide is administered to a mammal less frequently in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal using an otherwise equivalent molar amount, the factor IX polypeptide achieves a comparable therapeutic effect as the corresponding factor IX that lacks the XTEN;

(vi) when an accumulatively smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose period, the factor IX polypeptide achieves comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN; or

(vii) when an accumulatively smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose period, the factor IX polypeptide achieves comparable therapeutic effect as the corresponding factor IX that lacks the XTEN.

14. A method of treating coagulopathy in a subject, comprising administering to said subject a composition comprising a therapeutically effective amount of the factor IX polypeptide of any ofclaim 1.

15. The method ofclaim 14, wherein said coagulopathy is hemophilia B.

16. A method of treating a bleeding episode in a subject comprising administering to said subject a composition comprising a therapeutically effective amount of the factor IX polypeptide ofclaim 1.

17. A method of treating a subject deficient in a clotting protein, comprisings: administering to said subject a composition comprising a therapeutically effective amount of the factor IX of any ofclaim 1.

18. The method ofclaim 17, wherein the clotting protein substitutes wildetype factor VII, factor IX, or factor XI.

Images