Methods and compositions for DNA base editing

Abstract

The present invention relates to methods and compositions for modifying target sites in the genome of a cell. Fusion proteins are provided, which include one or more DNA binding domains and one or more heterologous domains, such as DNA modification domains, connected by improved linker sequences. Codon-optimized polynucleotides are provided, which encode fusion proteins including one or more DNA binding domains and one or more heterologous domains connected by improved linker sequences.

Description

Technical Field

[0001] The present invention relates to methods and compositions for targeted nucleotide base editing in the genome of a cell.

[0002] Statement Regarding Electronic Submission of Sequence Listings

[0003] Attached herewith and archived is a Sequence Listing in ASCII text format, filed pursuant to 37 CFR §1.821, entitled “81945_ST25,” created on September 18, 2020, approximately 702 kilobytes, and incorporated herein by reference. Background Art

[0004] The ability to edit plant genomes in order to create favorable alleles is highly desirable in agriculture. This could increase yields or prevent disease. Genome editing is a new field, with progress in plants lagging behind. Furthermore, in addition to the expected changes, changes in the genome are a problem that limits the realization of the desired changes. CRISPR-CAS9 works by double-stranded DNA cleavage. Since this break is repaired by non-homologous end joining or homology-dependent repair, DNA base insertions or deletions may occur. A strategy called base editing can alter DNA without cutting and producing insertions and deletions. In one version, an enzyme called cytidine deaminase targets specific bases through CAS9 (Shimatani et al., 2017. Nat. Biotechnol. [Nature Biotechnology] 35, 441-443) or CAS12a (Li et al., 2018. Nat. Biotechnol. [Nature Biotechnology] 36, 324-327) enzymes, and the CAS9 or CAS12a enzymes are modified to prevent them from cutting DNA. Cytidine deaminase and nuclease-deficient CAS9 or CAS12a are fused together via an amino acid linker. Improvements in linker ligation can improve the functionality of the fusion protein, such as improving the accuracy of cleavage by reducing changes in target bases. Summary of the Invention

[0005] In order to meet this need for improvement, we provide optimized and improved Cas12a enzymes and constructs. In particular, we provide a fusion protein comprising a heterologous domain, a first linker sequence, and a V-type CRISPR-Cas enzyme. The first linker sequence comprises a repeated GGGGS sequence. The heterologous domain can be a deaminase, a polymerase, a nuclease, a relaxase, an alkyltransferase, a methyltransferase, adenosine deaminase, a cytidine deaminase, an oxidase, a thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, a glycosylase, or a nuclear localization signal. For base editing, the heterologous domain is a deaminase domain such as a cytidine deaminase or adenine deaminase. The cytidine deaminase domain can be an activation-induced cytidine deaminase ("AID") or apolipoprotein B mRNA-editing complex ("APOBEC") domain, such as the APOBEC1 family from a deaminase. In some contexts, the APOBEC domain comprises a sequence having at least 70% identity to SEQ ID NO:1. Where an adenine deaminase is desired, the adenine deaminase may be a TadA domain comprising an amino acid sequence at least 70% identical to SEQ ID NO:92.

[0006] When the V-type CRISPR-Cas enzyme is a V-type-A ("Cas12a") enzyme, Cas12a is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. The Cas12a domain can be catalytically inactive but still binds to the target DNA and allows the heterologous domain to function. When Cas12a is inactive, its sequence is SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 22.

[0007] The first linker sequence between the heterologous domain and the Cas12a enzyme can include GGGGS repeated at least three times. In other uses, the first linker sequence can include GGGGS repeated at least six times.

[0008] The fusion protein can include SEQ ID NO: 11, 12, 13 or 44, and it can also include a uracil DNA glycosylase inhibitor ("UGI") domain (as represented by SEQ ID NO: 8). The UGI domain can be connected to the Cas12a enzyme by a second joint comprising the sequence SGGS. The fusion protein can include SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89. When contacted with DNA, these fusion proteins produce on-target editing with an increased frequency compared to the prior art fusion proteins lacking the first joint sequence of the repeated GGGGS sequence, and produce off-target editing with a reduced frequency.

[0009] We also provide a method for editing plant genomic DNA by contacting the plant genomic DNA with: (a) a fusion protein as described in one of the above aspects, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target edits compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0010] We also provide a method for editing plant genomic DNA with reduced off-target editing, the method comprising contacting the plant genomic DNA with: (a) a fusion protein as described in one of the above aspects, and the fusion protein optionally comprising a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the fusion protein comprises SEQ ID NO: 24.

[0011] We also provide a method for obtaining an edited plant population with reduced off-target editing by: (a) obtaining a population of plant cells comprising genomic DNA to be edited; (b) obtaining a nucleotide sequence encoding a fusion protein as described by one of the above aspects, and optionally a UGI domain; (c) transforming the population of plant cells with the nucleotide sequence of step (b), thereby expressing the fusion protein encoded by the nucleic acid sequence within the population of plant cells; (d) growing the population of transformed plant cells into plants, wherein at least one plant is edited; and (e) selecting at least one edited plant from the products of step (d), thereby obtaining an edited plant population; wherein the edited plant population comprises reduced off-target editing compared to plants edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, or SEQ ID NO: 89. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows a schematic diagram of the DNA construct of three versions of Cas12aBE. (1) represents a promoter; (2) is a nuclear localization signal; (3) is a deaminase, for example, an APOBEC deaminase; (4) is an XTEN linker; (5) is LbCas12a; (6) is an SGGS linker; (7) is a uracil glycosylase inhibitor; (8) is a long linker, for example, a (G4S)6 linker; (9) is Mb2Cas12a; (10) is an element encoding a guide RNA. Figure 1A shows an LbCas12aBE plus guide RNA construct in a 5' to 3' direction, wherein the deaminase (3) is operably connected to the LbCas12a (5) through the XTEN linker (4). Figure 1B shows an LbCas12aBE plus guide RNA construct in a 5' to 3' direction, wherein the deaminase (3) is operably connected to the LbCas12a (5) through the (G4S)6 linker (8). Figure 1C shows the Mb2Cas12aBE plus guide RNA construct in a 5' to 3' orientation, where the deaminase (3) is operably linked to the Mb2Cas12a (9) via a (G4S)6 linker (8).

[0013] Figure 2 shows a schematic diagram of a DNA construct in the 5' to 3' direction, which includes Cas12aBE and multiple guide RNAs. (1) represents a promoter; (2) is a nuclear localization signal; (3) is a deaminase, for example, an APOBEC deaminase; (6) is an SGGS linker; (7) is a uracil glycosylase inhibitor; (8) is a long linker, for example, a (G4S)6 linker; (9) is Cas12a; (10) is an element encoding a first guide RNA; (11) is an element encoding a second guide RNA; and (12) is an element encoding a third guide RNA. Each element encoding a guide RNA includes a crRNA segment and a target sequence segment capable of hybridizing with a genomic target DNA sequence.

[0014] Brief description of the sequences in the sequence listing

[0015] SEQ ID NO: 1 is the amino acid sequence of Apobec1.

[0016] SEQ ID NO: 2 is the nucleotide sequence of Apobec1.

[0017] SEQ ID NO: 3 is the amino acid sequence of catalytically inactive Mb2Cas12a.

[0018] SEQ ID NO: 4 is the nucleotide sequence of catalytically inactive Mb2Cas12a.

[0019] SEQ ID NO: 5 is the nucleotide sequence of catalytically inactive cLbCas12aBE.

[0020] SEQ ID NO: 6 is the amino acid sequence of catalytically inactive cLbCas12aBE.

[0021] SEQ ID NO: 7 is the nucleotide sequence of uracil DNA glycosylase inhibitor (UGI).

[0022] SEQ ID NO: 8 is the amino acid sequence of uracil DNA glycosylase inhibitor (UGI).

[0023] SEQ ID NO: 9 is a nucleotide sequence comprising the expression cassette prSoUbi4:SV40NLS:cLbCas12aBE:GS6 linker:SV40NLS:SGGS linker:UGI:SGGS linker:SV40NLS:tNOS.

[0024] SEQ ID NO: 10 is the nucleotide sequence of the optimized (G4S)x6 linker.

[0025] SEQ ID NO: 11 is the amino acid sequence of the optimized (G4S)x6 linker.

[0026] SEQ ID NO: 12 is the amino acid sequence of the 18 aa linker-SX.

[0027] SEQ ID NO: 13 is the amino acid sequence of the 15 aa linker-(G4S)X3.

[0028] SEQ ID NO: 14 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-07 from construct 25057.

[0029] SEQ ID NO: 15 is the amino acid sequence comprising the fusion protein cLBCas12aBE-07 from construct 25057.

[0030] SEQ ID NO: 16 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-08 from construct 25058.

[0031] SEQ ID NO: 17 is the amino acid sequence comprising the fusion protein cLBCas12aBE-08 from construct 25058.

[0032] SEQ ID NO: 18 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-01 from construct 24524.

[0033] SEQ ID NO: 19 is the amino acid sequence comprising the fusion protein cLBCas12aBE-01 from construct 24524.

[0034] SEQ ID NO: 20 is the nucleotide sequence of cCas9BE-02.

[0035] SEQ ID NO: 21 is the amino acid sequence of cCas9BE-02.

[0036] SEQ ID NO: 22 is the amino acid sequence of catalytically inactive AsCas12a.

[0037] SEQ ID NO: 23 is a nucleotide sequence comprising the fusion protein cLBCas12aBE-06 from construct 24904.

[0038] SEQ ID NO: 24 is the amino acid sequence comprising the fusion protein cLBCas12aBE-06 from construct 24904.

[0039] SEQ ID NO: 25 is a nucleotide sequence comprising the promoter prSoUbi4-02.

[0040] SEQ ID NO: 26 is a nucleotide sequence comprising the Cas12a gRNA waxy1 target sequence.

[0041] SEQ ID NO: 27 is a nucleotide sequence comprising the Cas9 gRNA waxy1 target sequence.

[0042] SEQ ID NO: 28 is a nucleotide sequence comprising exon 4 of the ZmWaxy1 gene.

[0043] SEQ ID NO: 29 is the forward primer of ZmWaxy1.

[0044] SEQ ID NO: 30 is the reverse primer of ZmWaxy1.

[0045] SEQ ID NO: 31 is the sequencing primer of ZmWaxy1.

[0046] SEQ ID NO: 32 is a nucleotide sequence comprising the fusion protein cLbCpf1-02 from construct 24523.

[0047] SEQ ID NO: 33 is the amino acid sequence comprising the fusion protein cLbCpf1-02 from construct 24523.

[0048] SEQ ID NO: 34 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25181.

[0049] SEQ ID NO: 35 is the amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25181.

[0050] SEQ ID NO: 36 is a nucleotide sequence comprising the fusion protein cLbCas12a-02 from construct 25205.

[0051] SEQ ID NO: 37 is the amino acid sequence comprising the fusion protein cLbCas12a-02 from construct 25205.

[0052] SEQ ID NO: 38 is a nucleotide sequence comprising the fusion protein cLbCas12a-25 from construct 25513.

[0053] SEQ ID NO: 39 is the amino acid sequence comprising the fusion protein cLbCas12a-25 from construct 25513.

[0054] SEQ ID NO: 40 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-01 from construct 25220.

[0055] SEQ ID NO: 41 is the amino acid sequence comprising the fusion protein cMb2Cas12a-01 from construct 25220.

[0056] SEQ ID NO: 42 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-02 from construct 25382.

[0057] SEQ ID NO: 43 is the amino acid sequence comprising the fusion protein cMb2Cas12a-02 from construct 25382.

[0058] SEQ ID NO: 44 is the amino acid sequence of the optimized (G4SG)x6 linker.

[0059] SEQ ID NO: 45 is the amino acid sequence of active LbCas12a.

[0060] SEQ ID NO: 46 is the amino acid sequence of active Mb2Cas12a.

[0061] SEQ ID NO: 47 is the amino acid sequence of active AsCas12a.

[0062] SEQ ID NO: 48 is the amino acid sequence of active FnCas12a.

[0063] SEQ ID NO: 49 is a nucleotide sequence comprising the fusion protein cMb2Cas12a-BE-01 from construct 25457.

[0064] SEQ ID NO: 50 is the amino acid sequence comprising the fusion protein cMb2Cas12a-BE-01 from construct 25457.

[0065] SEQ ID NO: 51 is a nucleotide sequence comprising the fusion protein cLbCas12a-BE-08 from construct 25268.

[0066] SEQ ID NO: 52 is the amino acid sequence comprising the fusion protein cLbCas12a-BE-08 from construct 25268.

[0067] SEQ ID NO: 53 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25173.

[0068] SEQ ID NO: 54 is the amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25173.

[0069] SEQ ID NO: 55 is a nucleotide sequence comprising the fusion protein cLbCas12a-05 from construct 25175.

[0070] SEQ ID NO: 56 is the amino acid sequence comprising the fusion protein cLbCas12a-05 from construct 25175.

[0071] SEQ ID NO: 57 is the amino acid sequence of a catalytically inactive LbCas12a with an optimized (G4SG)6 linker.

[0072] SEQ ID NO: 58 is the amino acid sequence of active Mb2Cas12a with an optimized (G4S)6 linker.

[0073] SEQ ID NO: 59 is the amino acid sequence of catalytically inactive Mb2Cas12a with an XTEN linker.

[0074] SEQ ID NO: 60 is the amino acid sequence of active AsCas12a with an XTEN linker.

[0075] SEQ ID NO: 61 is the amino acid sequence of catalytically inactive AsCas12a with an XTEN linker.

[0076] SEQ ID NO: 62 is the amino acid sequence of active FnCas12a with an XTEN linker.

[0077] SEQ ID NO: 63 is the amino acid sequence of active AsCas12a with an optimized (G4S)6 linker.

[0078] SEQ ID NO: 64 is the amino acid sequence of a catalytically inactive AsCas12a with an optimized (G4S)6 linker.

[0079] SEQ ID NO: 65 is the amino acid sequence of active FnCas12a with an optimized (G4S)6 linker.

[0080] SEQ ID NO: 66 is the amino acid sequence of a catalytically inactive Mb2Cas12a with an optimized (G4SG)6 linker.

[0081] SEQ ID NO: 67 is the amino acid sequence of active AsCas12a with an optimized (G4SG)6 linker.

[0082] SEQ ID NO: 68 is the amino acid sequence of a catalytically inactive AsCas12a with an optimized (G4SG)6 linker.

[0083] SEQ ID NO: 69 is the amino acid sequence of active FnCas12a with an optimized (G4SG)6 linker.

[0084] SEQ ID NO: 70 is the amino acid sequence of the XTEN linker.

[0085] SEQ ID NO: 71 is a nucleotide sequence comprising the Cas12a gRNA SBEII target sequence.

[0086] SEQ ID NO:72 is a nucleotide sequence comprising the Cas12a gRNA GL2 target sequence.

[0087] SEQ ID NO:73 is a nucleotide sequence comprising the Cas12a gRNA Fad2 target sequence.

[0088] SEQ ID NO: 74 is a nucleotide sequence comprising a Cas12a crRNA sequence for use with waxy1, SBEII, and Fad2 target sequences.

[0089] SEQ ID NO: 75 is a nucleotide sequence comprising a Cas12 acrRNA sequence for use with a GL2 target sequence.

[0090] SEQ ID NO:76 is a nucleotide sequence comprising the fusion protein cCas9ABE-01 from construct 24785.

[0091] SEQ ID NO: 77 is the amino acid sequence comprising the fusion protein cCas9ABE-01 from construct 24785.

[0092] SEQ ID NO: 78 is a nucleotide sequence comprising the fusion protein cLbCas1aABE-01 from construct 25459.

[0093] SEQ ID NO: 79 is the amino acid sequence comprising the fusion protein cLbCas1aABE-01 from construct 25459.

[0094] SEQ ID NO: 80 is a nucleotide sequence comprising the fusion protein cLbCas12aABE-02 from construct 25504.

[0095] SEQ ID NO: 81 is the amino acid sequence comprising the fusion protein cLbCas12aABE-02 from construct 25504.

[0096] SEQ ID NO: 82 is a nucleotide sequence comprising the fusion protein cLbCas12aBE-09 from construct 25289.

[0097] SEQ ID NO: 83 is the amino acid sequence comprising the fusion protein cLbCas12aBE-09 from construct 25289.

[0098] SEQ ID NO: 84 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-01 from construct 25658.

[0099] SEQ ID NO: 85 is the amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-01 from construct 25658.

[0100] SEQ ID NO: 86 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-02 from construct 25701.

[0101] SEQ ID NO: 87 is the amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-02 from construct 25701.

[0102] SEQ ID NO: 88 is a nucleotide sequence comprising the fusion protein cdLbCas12a-ABE-CBE-03 from construct 25702.

[0103] SEQ ID NO: 89 is the amino acid sequence comprising the fusion protein cdLbCas12a-ABE-CBE-03 from construct 25702.

[0104] SEQ ID NO: 90 is a nucleotide sequence comprising the Cas12a gRNA ADH1 target sequence.

[0105] SEQ ID NO: 91 is a nucleotide sequence comprising a TadA dimer.

[0106] SEQ ID NO: 92 is an amino acid sequence comprising a TadA dimer. DETAILED DESCRIPTION

[0107] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented, or all the features that may be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Furthermore, in light of this disclosure, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the present invention. Accordingly, the following description is intended to illustrate some specific embodiments of the present invention and is not intended to be an exhaustive description of all permutations, combinations, and variations thereof.

[0108] definition

[0109] 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. The terms used in the description of the invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0110] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in the practice of the present invention. Unless otherwise indicated, the terms used herein should be understood according to conventional usage by those skilled in the relevant art. Definitions of general terms in molecular biology can also be found in Rieger et al., Glossary of Genetics: Classical and Molecular Glossary of Genetics: Standard and Molecular, 5th ed., Springer-Verlag, New York, 1994.

[0111] As used herein, the term "long linker" refers to a polypeptide chain of at least 10 amino acids for connecting a heterologous domain to a protein of interest. By way of example and not limitation, a long linker can include the sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 11), additionally expressed as (G4S)6 or (G4S)x6 or (G4S)*6. A long linker can include GGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSG (SEQ ID NO: 44), additionally expressed as (G4SG)6 or (G4SG)x6 or (G4SG)*6. The heterologous domain connected to the protein by a long linker includes cytidine deaminase, guanine deaminase, uracil glycosylase inhibitor ("UGI"), nuclease, and any other protein domain that can be operably connected to the protein of interest in a heterologous manner. Such proteins of interest include, but are not limited to, site-directed nucleases (e.g., Cas9, Cas12a, Cas12b, Cas12i, Cas12j, or other CRISPR nucleases), zinc fingers, homing endonucleases, transcription activator-like effector nucleases ("TALENs"), and the like.

[0112] As used in the description of embodiments of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0113] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0114] As used herein, the term "about" when referring to a measurable value such as an amount of a compound, dosage, time, temperature, etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0115] The terms “comprise”, “comprises” and / or “comprising” when used in this specification specify the presence of listed features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0116] As used herein, the transition phrase "consisting essentially of means that the scope of a claim will be interpreted to encompass the specified materials or steps recited in the claim as well as those materials or steps that do not materially affect one or more of the basic and novel characteristics of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising."

[0117] As used herein, the term "amplified" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. See, for example, Diagnostic Molecular Microbiology: Principles and Applications, D.H. PERSING et al., eds., American Society for Microbiology, Washington, DC (1993). The amplified product is called an amplicon.

[0118] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (e.g., mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In some embodiments, the RNA is subsequently translated in vivo to produce a protein.

[0119] As used herein, the term "event" of transgenic refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising one or more target genes (e.g., transgenic)). The term "event" refers to the original transformant comprising heterologous DNA and / or the progeny of this transformant. The term "event" also refers to the progeny produced by sexual outcrossing (outcross) between a transformant and another strain. Even after repeated backcrossing to the recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present in the same chromosome position of the hybrid progeny. Typically, the transformation of plant tissue produces multiple events, and each of the above-mentioned events represents that a DNA construct is inserted into the different positions in the genome of the plant cell. Based on the expression of transgenic or other desired characteristics, a specific event is selected. Thus, as used herein, "event MIR604," "MIR604," or "MIR604 event" refers to the original MIR604 transformant and / or progeny of a MIR604 transformant (U.S. Patent Nos. 7,361,813; 7,897,748; 8,354,519 and 8,884,102, incorporated herein by reference).

[0120] As used herein, "expression cassette" means a nucleic acid molecule capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, the nucleic acid molecule comprising a promoter operably linked to a target nucleotide sequence (typically a coding region), the nucleotide sequence being operably linked to a termination signal. It also typically comprises the sequence required for the appropriate translation of the nucleotide sequence. The coding region typically encodes the target protein, but can also encode a functional RNA of interest (e.g., antisense RNA or non-translated RNA) in a sense or antisense direction. The expression cassette can also be included in sequences that are not required for directing the expression of the target nucleotide sequence, but it exists because of convenient restriction sites for removing the expression cassette from the expression vector. The expression cassette comprising the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette can also be naturally occurring but has been an expression cassette obtained in a recombinant form useful for heterologous expression. However, typically the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally occurring in the host cell and must have been introduced into the host cell or the ancestor of the host cell by transformation methods known in the art. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms (such as plants), the promoter can also be specific for a particular tissue, organ, or developmental stage. When transformed into a plant, the expression cassette or fragment thereof can also be referred to as an "inserted sequence" or "insertion sequence."

[0121] "Gene" is a limited area located within the genome, and in addition to the aforementioned coding nucleic acid sequence, it also includes other major regulatory nucleic acid sequences responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). A gene typically expresses mRNA, functional RNA, or specific protein, including regulatory sequences. A gene may or may not be used to produce a functional protein. In certain embodiments, a gene refers only to a coding region. The term "natural gene" refers to a gene as found in nature. The term "chimeric gene" refers to any gene comprising the following: 1) a DNA sequence, including regulatory sequences and coding sequences not found together in nature, or 2) a sequence encoding a portion of an unnaturally adjacent protein, or 3) a portion of an unnaturally adjacent promoter. Therefore, a chimeric gene can include regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged in a different manner from those found in nature. A gene can be "isolated," which means a nucleic acid molecule that is substantially (or essentially) free of components normally found associated with the nucleic acid molecule in its natural state. Such components include other cellular materials, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acid molecules.

[0122] The terms "express" or "expression" with reference to a polynucleotide coding sequence mean that the sequence is transcribed, and optionally translated.

[0123] "Gene of interest" or "nucleotide sequence of interest" refers to any gene that, when transferred to a plant, confers a desired characteristic on the plant (e.g., antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance of an industrial process, or altered reproductive capacity). A "gene of interest" may also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0124] As used herein, "heterologous" refers to a nucleic acid molecule or nucleotide sequence that is naturally unrelated to the host cell to which it is introduced, and the sequence is derived from another species or from the same species or organism, but has been modified from its original form or the form primarily expressed in the cell, including multiple copies of the non-naturally occurring nucleic acid sequence. Therefore, a nucleotide sequence derived from an organism or species different from the organism or species to which the cell to which it is introduced belongs is heterologous relative to that cell or the progeny of the cell. In addition, a heterologous nucleotide sequence includes a nucleotide sequence that is derived from and inserted into the same natural original cell type, but exists in a non-natural state, for example, exists in different copy numbers, and / or is under the control of regulatory sequences different from those found in the natural state of the nucleic acid molecule. The nucleic acid sequence can also be heterologous to other nucleic acid sequences associated therewith, for example, in a nucleic acid construct, such as, for example, an expression vector. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that do not naturally exist in relation to that particular promoter, i.e., they are heterologous to the promoter.

[0125] A "homologous" nucleic acid sequence is a nucleic acid sequence that is naturally associated with the host cell into which it is introduced. Homologous nucleic acid sequences can also be nucleic acid sequences naturally associated with other nucleic acid sequences, which can, for example, be present in a nucleic acid construct. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that naturally occur in association with that particular promoter, i.e., they are homologous to the promoter.

[0126] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid sequence so that the function of one affects the function of another. For example, when a promoter can affect the expression of a coding sequence or a functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter), the promoter is operably linked to the coding sequence or functional RNA. A coding sequence in the sense or antisense direction can be operably linked to a regulatory sequence. Therefore, a regulatory or control sequence (e.g., a promoter) that is operably associated with a nucleotide sequence can affect the expression of the nucleotide sequence. For example, a promoter that is operably linked to a nucleotide sequence encoding GFP will be able to affect the expression of the GFP nucleotide sequence.

[0127] Control sequences need not be contiguous to the nucleotide sequence of interest, as long as they function to direct its expression. Thus, for example, intervening untranslated, transcribed sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0128] As used herein, "primers" are isolated nucleic acids that are annealed to complementary target DNA strands by nucleic acid hybridization to form hybrids between the primers and the target DNA strands, which are then extended along the target DNA strands by a polymerase (e.g., DNA polymerase). Primer pairs or primer sets can be used for amplification of nucleic acid molecules, for example, by polymerase chain reaction (PCR) or other nucleic acid amplification methods.

[0129] " probe " is the nucleic acid molecule of the separation that is complementary to a part of target nucleic acid molecule, and is typically used for detecting and / or quantitative target nucleic acid molecule.Therefore, in some embodiments, probe can be the nucleic acid molecule of the separation that detectable part or reporter gene are attached to, as radioisotope, part, chemiluminescent agent, fluorescent agent or enzyme.Probe according to the present invention can not only comprise deoxyribonucleic acid or ribonucleic acid, also comprise and be combined with target nucleic acid sequence specificity and can be used for detecting the existence of target nucleic acid sequence or polyamides and other probe materials of the amount of quantitative target nucleic acid sequence.

[0130] The TaqMan probe is designed so that it anneals within the DNA region amplified by a specific primer set. Since the Taq polymerase extends the primer and synthesizes a nascent chain from the 3' to 5' single-stranded template of the complementary chain, the 5' to 3' exonuclease of the polymerase extends the nascent chain through the probe and thus degrades the probe that has annealed to the template. The degradation of the probe releases the fluorophore therefrom and breaks the close contact with the quencher, thereby reducing the quenching effect and allowing the fluorescence of the fluorophore. Therefore, the fluorescence detected in the quantitative PCR thermal cycler is proportional to the amount of released fluorophore and the DNA template present in the PCR.

[0131] The length of primer and probe is generally between 5 and 100 Nucleotide or more Nucleotide.In certain embodiments, the length of primer and probe can be at least 20 Nucleotide or more, or at least 25 Nucleotide or more, or length at least 30 Nucleotide or more.These primers and probes are under optimal hybridization condition known in the art and target sequence specific hybridization.Can have the complete sequence complementary with target sequence according to primer and probe of the present invention, though different from target sequence and keep the probe with the ability of target sequence hybridization by designing according to ordinary method of the present invention.

[0132] Methods for making and using probes and primers are described, e.g., Molecular Cloning:A Laboratory Manual Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. PCR primer pairs can be derived from known sequences, for example, by using computer programs designed for this purpose.

[0133] Polymerase chain reaction (PCR) is a technique for "amplifying" specific DNA fragments. In order to perform PCR, at least a portion of the nucleotide sequence of the DNA molecule to be replicated must be known. Typically, primers or short oligonucleotides that are complementary (e.g., substantially complementary or completely complementary) to the nucleotide sequence (known sequence) at the 3' end of each chain of the DNA to be amplified are used. The DNA sample is heated to separate its chains and mixed with these primers. These primers hybridize with the complementary sequences in their DNA sample. Synthesis (5' to 3' direction) is started using the original DNA chain as a template. The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) and DNA polymerase. Polymerization continues until each newly synthesized chain has progressed far enough to contain a sequence recognized by another primer. Once this occurs, two DNA molecules identical to the original molecule are produced. The two molecules are heated to separate their chains, and the process is repeated. Each cycle doubles the number of DNA molecules. Using automated equipment, replication of each cycle can be completed in less than 5 minutes. After 30 cycles, the amplification starting from a single DNA molecule has exceeded 1 billion copies (2 30 =1.02x 10 9 ).

[0134] The oligonucleotides of the oligonucleotide primer pair are complementary to the DNA sequence located on the relative DNA chain and flanking the region to be amplified. The annealed primers hybridize with the newly synthesized DNA chain. The first amplification cycle will result in two new DNA chains, whose 5' ends are fixed by the position of the oligonucleotide primers, but whose 3' ends are variable ('irregular' 3' ends). The two new chains can serve as templates for the synthesis of complementary chains of desired length in turn (the 5' end is defined by the primer and the 3' end is fixed because the synthesis can not exceed the end of the opposite primer). After several cycles, the desired fixed-length product begins to dominate.

[0135] Quantitative polymerase chain reaction (qPCR) (also referred to as real-time polymerase chain reaction) monitors the accumulation of DNA products from PCR reactions in real time. qPCR is a molecular biology laboratory technique based on polymerase chain reaction (PCR) for amplifying and simultaneously quantifying target DNA molecules. Even one copy of a specific sequence can be amplified and detected in PCR. The PCR reaction generates copies of the DNA template in an exponential manner. This results in a quantitative relationship between the amount of the starting target sequence and the amount of PCR product accumulated under any particular cycle. Due to inhibitors of the polymerase reaction found together with the accumulation of template, reagent limitations, or pyrophosphate molecules, the PCR reaction eventually stops generating templates at an exponential rate (i.e., plateau phase), making the endpoint quantification of the PCR product unreliable. Therefore, repeated reactions can generate variable amounts of PCR products. Only during the exponential phase of the PCR reaction is it possible to push back to determine the starting amount of the template sequence. Measurement during PCR product accumulation (i.e., real-time quantitative PCR) allows for quantification during the exponential phase of the reaction, and therefore eliminates the variability associated with conventional PCR. In real-time PCR assays, positive reactions are detected by accumulation of fluorescent signals. Quantitative PCR can detect and quantify one or more specific sequences in a DNA sample. Quantity can be the absolute number of copies or the relative amount when normalized to DNA input or an additional normalizing gene. Since the first record of real-time PCR, it has been used for an increasing number of applications, including mRNA expression studies, DNA copy number measurements in genomes or viral DNA, allelic discrimination assays, expression analysis of specific splice variants of genes, and gene expression in paraffin-embedded tissues and laser-captured microdissected cells.

[0136] As used herein, the phrase "Ct value" refers to the "cycle threshold value", which is defined as "the fractional cycle number at which the amount of amplified target reaches a fixed threshold value". In some embodiments, it represents the intersection between the amplification curve and the threshold line. The amplification curve is typically in an "S" shape, which represents the change in relative fluorescence of each reaction (Y axis) at a given cycle (X axis), which is recorded during PCR by a real-time PCR instrument in some embodiments. In some embodiments, the threshold line is the detection level at which the reaction reaches a fluorescence intensity above the background. See Livak and Schmittgen (2001) 25 Methods [Methods] 402-408. It is a relative measure of the target concentration in PCR. Typically, in some embodiments, for a given reference gene, a good Ct value for quantitative determinations such as qPCR is in the range of 10-40. The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the higher the amount of detectable target nucleic acid in the sample). In addition, a good Ct value for quantitative determinations such as qPCR shows a linear response range when the target gDNA is proportionally diluted.

[0137] In some embodiments, qPCR is performed under conditions where Ct values ​​can be collected in real time for quantitative analysis. For example, in a typical qPCR experiment, DNA amplification is monitored at each cycle of PCR during the extension phase. When the DNA is in the log-linear phase of amplification, the amount of fluorescence typically increases above background. In some embodiments, Ct values ​​are collected at this time point.

[0138] As used herein, the term "cell" refers to any living cell. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be isolated. The cell may or may not be able to regenerate into an organism. The cell can be in the context of a tissue, callus, culture, organ, or part. In certain embodiments, the cell can be a plant cell. The plant cell of the present invention can be in an isolated unicellular form, or can be a cultured cell, or can be a part of a higher-level organizational unit (such as, for example, a plant tissue or a plant organ). The plant cell can be derived from angiosperms or gymnosperms or a part thereof. In other embodiments, the plant cell can be a monocotyledonous plant cell or a dicotyledonous plant cell. The monocotyledonous plant cell can be, for example, corn, rice, sorghum, sugarcane, barley, wheat, oats, turf grass, or an ornamental grass cell. The dicotyledonous plant cell can be, for example, a tobacco, pepper, eggplant, sunflower, cruciferous plant, flax, potato, cotton, soybean, sugar beet, or rapeseed cell.

[0139] As used herein, the term "plant part" includes, but is not limited to, embryos, pollen, ovules, seeds, leaves, stems, buds, flowers, branches, fruits, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells (including intact plant cells in plants and / or parts of plants), plant protoplasts, plant tissues, plant cell tissue cultures, plant callus, plant masses, and the like. As used herein, "bud" refers to the above-ground parts including leaves and stems. Additionally, as used herein, "plant cell" refers to the structural and physiological unit of a plant, including a cell wall and may also refer to a protoplast.

[0140] In the context of cells, prokaryotic cells, bacterial cells, eukaryotic cells, plant cells, plants and / or plant parts, the term "introducing" or "introduce" means contacting a nucleic acid molecule with a cell, eukaryotic cell, plant, plant part and / or plant cell in such a way that the nucleic acid molecule is able to enter the interior of the cell, eukaryotic cell, plant cell and / or plant and / or plant part. When more than one nucleic acid molecule is introduced, these nucleic acid molecules can be assembled into a part of a single polynucleotide or nucleic acid construct, or assembled into separate polynucleotides or nucleic acid constructs and can be located on the same or different nucleic acid constructs. Thus, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, for example, as part of a breeding program.

[0141] As used herein, the terms "transformed" and "transgenic" refer to any cell, prokaryotic cell, eukaryotic cell, plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide. In some embodiments, all or part of the recombinant polynucleotide is stably integrated into a chromosome or a stable extrachromosomal element so that it is passed on to successive generations. For the purposes of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or a polynucleotide that is linked or joined to a heterologous sequence. The term "recombinant" does not refer to polynucleotide alterations that occur due to naturally occurring events (e.g., spontaneous mutations) or due to non-spontaneous mutagenesis followed by selective breeding.

[0142] As used herein, the term "transformation" refers to the introduction of heterologous nucleic acid into a cell. The transformation of a cell can be stable or transient. Therefore, transgenic cells of the present invention, plant cells, plants and / or plant parts can be stably transformed or transiently transformed. The term "transformation" can refer to the transfer of nucleic acid molecules into the genome of a host cell, resulting in genetically stable inheritance. In certain embodiments, introducing into a plant, plant part and / or plant cell is via bacterial-mediated transformation, particle bombardment transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated transformation, protoplast transformation, or any other electrical, chemical, physical and / or biological mechanism that causes introducing nucleic acid into a plant, plant part and / or its cell, or any combination thereof.

[0143] The procedures for transforming plants are well known and conventional in the art and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacteria-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that allows the introduction of nucleic acids into plant cells, including any combination thereof. General guides to the various plant transformation methods known in the art include those by Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Plant Molecular Biology and Biotechnology, eds. Glick, BR and Thompson, JE (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0144] Agrobacterium-mediated transformation is a common method for transforming plants because of its high transformation efficiency and because of its wide applicability with many different species. Agrobacterium-mediated transformation typically involves transferring the binary vector carrying the exogenous DNA of interest to an appropriate Agrobacterium strain, which may depend on the complement of the vir genes carried by the host Agrobacterium strain on a co-existing Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell [plant cells] 5: 159-169). The Escherichia coli carrying the recombinant binary vector can be used, and the auxiliary Escherichia coli strain (the auxiliary Escherichia coli strain carries a plasmid that can move the recombinant binary vector into the target Agrobacterium strain) is passed through a three-parent mating procedure to achieve the transfer of the recombinant binary vector to Agrobacterium. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation ( and Willmitzer, 1988, Nucleic Acids Res. 16:9877).

[0145] Plant transformation by recombinant Agrobacterium typically involves the co-cultivation of Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is typically regenerated on selection media carrying an antibiotic or herbicide resistance marker located between the binary plasmid T-DNA borders.

[0146] Another method for transforming plants, plant parts, and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Typically, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide for incorporation into its interior. When inert particles are used, the vector can be introduced into the cell by coating these particles with a vector containing the target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is brought into the cell by stimulation of the particles. Biologically active particles (e.g., dried yeast cells, dried bacteria, or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissue.

[0147] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell and is not integrated into the genome of the cell.

[0148] As used herein, "stably introducing," "stably introduced," "stably transformed," or "stably transformed" in the context of a polynucleotide introduced into a cell means that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. Thus, the integrated polynucleotide can be inherited by its progeny, more specifically, by multiple successive generations of progeny. "Genome," as used herein, includes nuclear and / or plastid genomes, and therefore includes integration of a polynucleotide into, for example, a chloroplast genome. Stable transformation, as used herein, can also refer to a polynucleotide that is maintained extrachromosomally, for example, as a minichromosome.

[0149] Transient conversion can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or Western blotting, both of which can detect the presence of peptides or polypeptides encoded by one or more nucleic acid molecules introduced into an organism. Stable conversion of a cell can be detected by, for example, Southern blot hybridization assays of cellular genomic DNA and nucleic acid sequences (these sequences specifically hybridize to the nucleotide sequence of the nucleic acid molecules introduced into an organism (e.g., a plant). Stable conversion of a cell can be detected by, for example, Northern blot hybridization assays of cellular RNA and nucleic acid sequences (these sequences specifically hybridize to the nucleotide sequence of the nucleic acid molecules introduced into a plant or other organism). Stable conversion of a cell can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences hybridized to one or more target sequences of nucleic acid molecules, resulting in the amplification of one or more target sequences, which can be detected according to standard methods. Conversion can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0150] Thus, in particular embodiments of the present invention, plant cells can be transformed by any method known in the art and as described herein and whole plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture, and / or cultured protoplasts is described, for example, in Evans et al. ( Handbook of Plant Cell Cultures [Manual of Plant Cell Culture], Vol. 1, MacMilan Publishing Co., New York (1983); and Vasil IR (ed.) ( Cell Culture and Somatic Cell Genetics of Plants [Plant Cell Culture and Somatic Cell Genetics], Academic Press, Orlando, Volume I (Acad. Press, Orlando) (1984) and Volume II (1986). Methods for selecting transformed transgenic plants, plant cells and / or plant tissue cultures are routine in the art and can be used in the methods of the invention provided herein.

[0151] "Transformation and regeneration process" refers to the process of stably introducing a transgene into a plant cell and regenerating a plant from the transgenic plant cell. As used herein, transformation and regeneration include selection processes wherein the transgene comprises a selectable marker and the transformed cells have incorporated and express the transgene such that the transformed cells will survive and thrive in the presence of the selective agent. "Regeneration" refers to the growth of a whole plant from a plant cell, group of plant cells, or plant piece (e.g., from a protoplast, callus, or tissue portion).

[0152] The terms "nucleotide sequence," "nucleic acid," "nucleic acid sequence," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are used interchangeably herein to refer to heteropolymers of nucleotides and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term nucleic acid molecule refers to a chain of nucleotides, regardless of the length of the chain. These nucleotides comprise a sugar, a phosphate, and a base, which is a purine or a pyrimidine. Nucleic acid molecules can be double-stranded or single-stranded. When single-stranded, the nucleic acid molecule can be a sense strand or an antisense strand. Nucleic acid molecules can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can, for example, be used to prepare nucleic acid molecules with altered base pairing ability or enhanced resistance to nucleases. Nucleic acid sequences provided herein are represented herein from left to right in 5' to 3' orientation and are represented using the standard code for representing nucleotide characters as set forth in U.S. Sequence Rules, 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25.

[0153] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. An "RNA fragment" is a portion of a given RNA molecule. A "DNA fragment" is a portion of a given DNA molecule. A "nucleic acid segment" is a portion of a given nucleic acid molecule and is not isolated from that molecule. An "RNA segment" is a portion of a given RNA molecule and is not isolated from that molecule. A "DNA segment" is a portion of a given DNA molecule and is not isolated from that molecule. A segment of a polynucleotide can be of any length, e.g., at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length. A segment or portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10%, e.g., one-third of the guide sequence, or less, e.g., 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0154] In the context of molecules, the term "derived from" refers to a molecule that is isolated or made using a parent molecule or information from that parent molecule. For example, the Cas9 single mutant nickase and the Cas9 double mutant null nuclease are derived from the wild-type Cas9 protein.

[0155] In higher plants, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is involved in the transfer of the information contained in the DNA to the protein. A "genome" is the entirety of the genetic material contained in each cell of an organism. Unless otherwise indicated, the specific nucleic acid sequence of the present invention also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. [Nucleic Acids Research] 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. [Journal of Biological Chemistry] 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes [Molecular and Cellular Probes] 8: 91-98 (1994)). The term nucleic acid molecule is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0156] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire comparison window of components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology Sequence Analysis in Molecular Biology (von Heinje, G. ed.) Academic Press (1987); and Sequence Analysis Primer [Primers for Sequence Analysis] (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0157] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence.

[0158] As used herein, in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, the phrase "substantially identical" refers to two or more sequences or subsequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, there is substantial identity over a sequence region that is at least about 50 residues to about 150 residues in length. Thus, in some embodiments of the invention, there is substantial identity over a sequence region that is at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150 or more residues in length. In some specific embodiments, these sequences are substantially identical over at least about 150 residues. In further embodiments, the sequences are substantially identical over the entire length of the coding region. In addition, in representative embodiments, substantially identical nucleotide sequences or protein sequences perform substantially the same function (e.g., directing endonuclease cleavage to a specific genomic target, a specific genomic target site).

[0159] For sequence comparison, typically, one sequence acts as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer (if necessary, subsequence coordinates are specified), and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the specified program parameters.

[0160] Optimal sequence alignment for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as Wisconsin (Materials Science Software Company (Accelrys Inc., San Diego, CA) available as part of GAP, BESTFIT, FASTA and TFASTA. The "identity score" of the aligned segments of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., the complete reference sequence or a smaller defined portion of the reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. Comparison of one or more polynucleotide sequences can be relative to the full-length polynucleotide sequence or a portion thereof, or relative to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0161] The software for performing BLAST analysis can be publicly obtained through the National Center for Biotechnology Information (National Center for Biotechnology Information). This algorithm involves first identifying the sequence pairs (HSPs) with high scores by identifying short words with length W in the query sequence, and these high-scoring sequences match or meet the score T of some positive thresholds when compared with the word (word) with the same length in the database sequence. T is referred to as the contiguous word score threshold (Altschul et al., 1990). These initial contiguous words hit and serve as seeds for initial searches to find longer HSPs containing them. Then, these words are hit and extended along each sequence in both directions until the cumulative comparison score can be increased. For nucleotide sequences, parameters M (for a pair of matching residues reward score; always> 0) and N (for the penalty of mismatch residues; always< 0) are used to calculate cumulative scores. For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Extension of word hits in each direction is terminated when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score approaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0162] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.

[0163] When two nucleotide sequences hybridize to each other under stringent conditions, the two nucleotide sequences may also be considered to be substantially identical. In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under high stringency conditions.

[0164] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. A comprehensive guide to nucleic acid hybridization is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acids, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be 500 ng / min above the thermal melting point (Tf) for the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0165] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide with 1 mg of heparin at 42°C, where hybridization is performed overnight. An example of high stringency wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is washing at 65°C with 0.2x SSC for 15 minutes (see Sambrook, below, for a description of SSC buffer). Typically, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is washing at 45°C with 1x SSC for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is washing at 40°C with 4-6xSSC for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature typically of at least about 30° C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or higher) observed compared to an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. Nucleotide sequences that do not hybridize to each other under stringent conditions are still considered to be substantially identical if the proteins encoded by them are substantially identical. This may occur, for example, when a copy of a nucleotide sequence is generated using the maximum codon degeneracy allowed by the genetic code.

[0166] The following are examples of hybridization / wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention. In one embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, with a wash in 2X SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, with a wash in 1X SSC, 0.1% SDS at 50°C; or hybridized to 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, with a wash in 0.5X SSC, 0.1% SDS at 50°C. In still other embodiments, a reference nucleotide sequence is hybridized to a "test" nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA, with a wash at 50°C in 0.1X SSC, 0.1% SDS; or at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA, with a wash at 65°C in 0.1X SSC, 0.1% SDS.

[0167] "Isolated" nucleic acid molecules or nucleotide sequence or "isolated" polypeptide are nucleic acid molecules, nucleotide sequence or polypeptides that exist by artificially breaking away from their natural environment and / or have different, modified, regulated and / or changed functions and therefore are not natural products.Isolated nucleic acid molecules or isolated polypeptides can exist in purified form or can be present in non-natural environments (such as, for example, recombinant host cells).Therefore, for example, with respect to polynucleotides, the meaning of the term isolated is to separate polynucleotides from their naturally occurring chromosomes and / or cells therein. If a kind of polynucleotides is separated from its naturally occurring chromosomes and / or cells therein, and then inserted into a genetic background, chromosome, chromosomal position and / or cell therein that is not naturally present, then polynucleotides are also isolated.Recombinant nucleic acid molecules and nucleotide sequence of the present invention can be considered to as "isolated" as defined above.

[0168] Thus, an "isolated nucleic acid molecule" or "isolated nucleotide sequence" is a nucleic acid molecule or nucleotide sequence that is not adjacent to adjacent nucleotide sequences (either sequences located at the 5' end or sequences located at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately adjacent to the coding sequence. Thus, the term includes, for example, a recombinant nucleic acid that is incorporated into a vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment obtained by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid nucleic acid molecule that encodes another polypeptide or peptide sequence. An "isolated nucleic acid molecule" or "isolated nucleotide sequence" may also include a nucleotide sequence that is derived from and inserted into the same natural original cell type, but is present in a non-natural state, for example, in a different copy number, and / or under the control of regulatory sequences that are different from those found in the natural state of the nucleic acid molecule.

[0169] The term "isolated" can further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and / or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Additionally, an "isolated fragment" is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that does not naturally occur as a fragment and would not so exist in nature. "Isolated" does not necessarily mean that the preparation is industrially pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for its intended purpose.

[0170] In representative embodiments of the invention, "isolated" nucleic acid molecules, nucleotide sequences and / or polypeptides are at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w / w) or purer. In other embodiments, "isolated" nucleic acids, nucleotide sequences and / or polypeptides refer to at least about 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or greater enrichment (w / w) of nucleic acids compared to the starting material.

[0171] A "wild-type" nucleotide sequence or amino acid sequence refers to a naturally occurring ("native") or endogenous nucleotide sequence or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA naturally occurring in or endogenous to an organism. A "homologous" nucleotide sequence is a nucleotide sequence naturally associated with the host cell into which it is introduced.

[0172] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between the translation start and stop codons of a coding sequence. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides ('codons') in a coding sequence that specify initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0173] "Promoter" refers to a nucleotide sequence, usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing the recognition of the RNA polymerase and other factors required for appropriate transcription. "Promoter regulatory sequence" consists of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, non-translated leader sequences, introns, and polyadenylation signal sequences. They include natural sequences and synthetic sequences, and sequences that may be combinations of synthetic sequences and natural sequences. "Enhancer" is a DNA sequence that can stimulate the activity of the promoter and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. It can operate in two directions (normal or flipped) and can even play a role when moved upstream or downstream of the promoter. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0174] "Primary transformants" and "T0 generation" refer to transgenic plants of the same genetic generation as the tissue that was initially transformed (i.e., has not undergone meiosis and fertilization since transformation). "Secondary transformants" and "T1, T2, T3, etc. generations" refer to transgenic plants that have been derived from primary transformants through one or more cycles of meiosis and fertilization. They can be derived by self-fertilization of primary or secondary transformants or by crossing primary or secondary transformants with other transformed or untransformed plants.

[0175] "Transgenic" refers to a nucleic acid molecule that has been introduced into the genome by transformation and is stably maintained. A transgenic can comprise at least one expression cassette, typically at least two expression cassettes, and can comprise ten or more expression cassettes. A transgenic can include, for example, genes that are heterologous or homologous to the genes of the particular plant to be transformed. In addition, a transgenic can comprise a native gene that has been inserted into a non-natural organism, or a chimeric gene. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. An "exogenous" gene refers to a gene that is not normally found in the host organism but that has been introduced into the organism by gene transfer.

[0176] "Introns" refer to intervening segments of DNA that occur almost exclusively in eukaryotic genes but are not translated into amino acid sequences in the gene product. These introns are removed from immature mRNA by a process called splicing, which leaves the exons untouched, thereby forming the mRNA. For the purposes of the present invention, the definition of the term "intron" includes modifications to the nucleotide sequence derived from an intron of a target gene, provided that the modified intron does not significantly reduce the activity of its associated 5' regulatory sequence.

[0177] "Exon" refers to a segment of DNA that carries the coding sequence for a protein or a portion thereof. Exons are separated by intervening, non-coding sequences (introns). For the purposes of the present invention, the definition of the term "exon" includes modifications to the nucleotide sequence of an exon derived from a target gene, provided that the modified exon does not significantly reduce the activity of its associated 5' regulatory sequence.

[0178] The term "cleavage" or "cleaving" refers to the breakage of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" encompasses both single-strand breaks and double-strand breaks. As a result of two different single-strand cleavage events, double-strand cleavage can occur. Cutting can result in the production of blunt ends or staggered ends. A "nuclease cleavage site" or "genomic nuclease cleavage site" is a nucleotide region comprising a nuclease cleavage sequence that is recognized by a specific nuclease for cutting the nucleotide sequence of genomic DNA in one or both chains. This cutting by the nuclease triggers a DNA repair mechanism in the cell that establishes an environment in which homologous recombination occurs.

[0179] The present invention provides a fusion protein having an improved linker between a deaminase domain and a site-directed DNA-binding domain, providing increased editing efficiency and reduced mutation frequency. In some embodiments of the present invention, the deaminase domain is a cytidine deaminase. In other embodiments of the present invention, the deaminase domain is an adenine deaminase. In some embodiments, the cytidine deaminase domain is an activation-induced cytidine deaminase ("AID"). In some embodiments of the present invention, the cytidine deaminase domain is an apolipoprotein B mRNA-editing complex ("APOBEC") domain. In some embodiments, the APOBEC domain is an APOBEC1 family deaminase.

[0180] "Cytidine deaminase" refers to an enzyme that catalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to produce uridine and deoxyuridine, respectively. Cytidine deaminase maintains the cytosine pool. The family of cytidine deaminases is APOBEC ("Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like"). Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. RNA editing by APOBEC1 requires homodimerization, and the complex interacts with RNA-binding proteins to form an editing body. Non-limiting examples of APOBEC proteins include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. A variety of mutants of APOBEC proteins are also known, and these mutants bring different editing characteristics of base editors. For example, for human APOBEC3A, certain mutants (e.g., Y130F, Y132D, W104A, and D131Y) are even better than wild-type human APOBEC3A in terms of editing efficiency. Therefore, the term APOBEC and each of its family members also encompass variants and mutants that have a certain level (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) of sequence identity with the corresponding wild-type APOBEC protein and retain cytidine deamination activity. Variants and mutants can be obtained using amino acid additions, deletions, and / or substitutions. In some embodiments, such substitutions are conservative substitutions.

[0181] A "cytosine base editor" ("CBE") converts a C·G base pair into a T·A base pair.

[0182] "Adenine deaminase" refers to an enzyme that catalyzes the hydrolytic deamination of adenosine to produce inosine. Inosine pairs with a C and is therefore read or copied as a G. An exemplary enzyme is TadA from E. coli, which functions as a homodimer.

[0183] An "adenine base editor" ("ABE") converts A·T base pairs into G·C base pairs.

[0184] Lachnospiraceae bacterium Cpf1 (LbCpf1) is one of a large group of many Cpf1 proteins. The terms "Cpf1" and "Cas12a" are used interchangeably throughout. Cpf1 is a Cas protein. The term "Cas protein" or "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein" refers to RNA-guided DNA endonucleases associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-adaptive immune system found in, for example, Streptococcus pyogenes and other bacteria, including Cas9, Cas12a, Cas12b, Cas12i, Cas12j, etc. In some embodiments of the present invention, the site-directed DNA binding domain is a catalytically inactive Cas12a from Lachnospiraceae bacteria ("dLbCas12a"). In other embodiments, the site-directed DNA binding domain is catalytically active and is from a bacteria of the genus Lachnospiraceae ("LbCas12a") or Moraxella bovoculi AAX08_00205 ("Mb2Cas12a"). In some embodiments of the invention, a Cas12a protein from a bacteria of the genus Lachnospiraceae, a species of Acidinococcus, a species of Moraxella bovoculi, a species of Thiomicrospira sp., a species of Moraxella lacunata, Methanomethylophilus alvus, a species of Butyrivibrio sp., or a species of Bacteroidetesoral sp. is provided as a site-directed DNA-binding domain of a fusion protein.

[0185] The fusion protein may include other fragments, such as uracil DNA glycosylase inhibitor (UGI) and nuclear localization sequence (NLS).

[0186] "Uracil glycosylase inhibitors" (UGIs), which can be prepared from Bacillus subtilis phage PBS1, are small proteins (9.5 kDa) that inhibit Escherichia coli uracil-DNA glycosylase (UDG), as well as UDG from other species. Inhibition of UDG occurs by reversible protein binding with a 1:1 UGD:UGI stoichiometry. UGIs are capable of dissociating UDG-DNA complexes. A non-limiting example of a UGI is found in Bacillus phage AR9 (YP_009283008.1). In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 8 or has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 8, and retains uracil glycosylase inhibitory activity.

[0187] In some embodiments, the UGI is located on the C-terminal side of the cytidine deaminase-Cpf1 portion. In some embodiments, the fusion protein comprises at least two UGIs.

[0188] In some embodiments, at least one nuclear localization signal ("NLS") is located at the C-terminus of the first segment and the second segment (the cytidine deaminase-Cpf1 portion), e.g., between the second segment (including Cpf1) and the UGI. In some embodiments, at least two NLSs are located between the second segment and the UGI. In some embodiments, at least three NLSs are located between the second segment and the UGI. In some embodiments, at least one NLS is located at the N-terminus of the first segment and the second segment (the cytidine deaminase-Cpf1 portion).

[0189] Non-limiting example arrangements of components in the fusion protein include, from N-terminus to C-terminus, (a) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, and UGI; (b) NLS, cytidine deaminase, Cas12a, NLS, NLS, UGI, NLS, 2A, and UGI; (c) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, UGI, 2A, and UGI; (d) NLS, cytidine deaminase, Cas12a, NLS, UGI, NLS, 2A, UGI, 2A, UGI, 2A, and UGI.

[0190] In some embodiments, a peptide linker is optionally provided between each fragment in the fusion protein. In some embodiments, the peptide linker has 1 to 100 amino acid residues (or 3-20, 4-15, without limitation). In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the amino acid residues of the peptide linker are amino acid residues selected from the group consisting of: alanine, glycine, cysteine ​​and serine.

[0191] The present invention also provides nucleic acid molecules comprising nucleic acid sequences encoding guide RNAs of the present invention. The nucleic acid molecules can be DNA or RNA molecules. In some embodiments, the nucleic acid molecules are cyclized. In other embodiments, the nucleic acid molecules are linear. In some embodiments, the nucleic acid molecules are single-stranded, partially double-stranded, or double-stranded. In some embodiments, the nucleic acid molecules are complexed with at least one polypeptide. The polypeptide can have a nucleic acid recognition domain or a nucleic acid binding domain. In some embodiments, the polypeptide is used to mediate, for example, a shuttle for the delivery of a chimeric RNA of the present invention and optionally a nuclease. In some embodiments, the polypeptide is a Feldan shuttle (U.S. Patent Publication No. 20160298078, incorporated herein by reference).

[0192] "On-target editing" is the substitution of cytosine for thymine in the region following the PAM site targeted by the gRNA. The primary editing window is 8 to 13 bases after the PAM site. "Off-target editing" is an insertion or deletion (indel) or base change other than C to T within the gRNA target region, or a base change or insertion or deletion outside the gRNA target region.

[0193] "Site-directed modifying polypeptides" modify target DNA (e.g., cleavage or methylation of target DNA) and / or polypeptides associated with target DNA (e.g., methylation or acetylation of histone tails). Site-directed modifying polypeptides are also referred to herein as "site-directed polypeptides" or "RNA-binding site-directed modifying polypeptides." Due to the association of the site-directed modifying polypeptide with the guide RNA, the site-directed modifying polypeptide interacts with the guide RNA (the guide RNA is a single RNA molecule or an RNA duplex of at least two RNA molecules) and is guided to a DNA sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.).

[0194] In some cases, the site-directed modifying polypeptide is a naturally occurring modified polypeptide. In other cases, the site-directed modifying polypeptide is not a naturally occurring modified polypeptide (e.g., a chimeric polypeptide or a modified (e.g., mutated, deleted, inserted) naturally occurring polypeptide). Exemplary naturally occurring site-directed modifying polypeptides are known in the art (see, for example, Makarova et al., 2017, Cell 168:328-328.e1, and Shmakov et al., 2017, Nat Rev Microbiol 15(3):169-182, both of which are incorporated herein by reference). These naturally occurring polypeptides bind to DNA-targeting RNA and are thereby directed to a specific sequence within the target DNA and cut the target DNA, thereby generating a double-strand break.

[0195] The site-directed modifying polypeptide comprises two parts, namely an RNA binding portion and an active portion. In some embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding portion that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active portion that exhibits site-directed enzymatic activity (e.g., DNA methylation activity, DNA cleavage activity, histone acetylation activity, histone methylation activity, etc.), wherein the site of enzymatic activity is determined by the DNA-targeting RNA. In other embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding portion that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active portion that regulates transcription (e.g., increases or decreases transcription) in the target DNA, wherein the site of regulated transcription in the target DNA is determined by the DNA-targeting RNA.

[0196] In some cases, the site-directed modifying polypeptide has an operably connected heterologous domain. The heterologous domain can be an enzyme or a signal peptide. In aspects where the heterologous domain is an enzyme domain, the domain has an enzymatic activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deaminating activity, reverse transcriptase activity, dismutase activity, alkylation activity, methylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity) that modifies the target nucleic acid. In other cases, the site-directed modifying polypeptide has an operably linked enzyme domain whose enzymatic activity modifies a polypeptide (e.g., a histone) associated with the target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Exemplary enzyme domains include adenosine deaminase, oxidase, thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, adenosine deaminase, glycosylase, either alone or in combination with other enzyme domains. In aspects where the heterologous domain is a signal peptide, the signal peptide can be a nuclear localization signal ("NLS"), such as the SV40 NLS.

[0197] In some cases, different site-directed modifying polypeptides, such as different Cas9 proteins (i.e., Cas9 proteins from various species), can be advantageously used in a variety of methods provided herein to utilize a variety of enzymatic features of different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for increased or decreased cytotoxicity levels; for changing the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.). Cas9 proteins from various species (e.g., those disclosed in Shmakov et al., 2017, or polypeptides derived therefrom) may require different PAM sequences in the target DNA. Therefore, for the selection of a specific Cas9 enzyme, the PAM sequence requirement may be different from the 5'-NGG-3' sequence (wherein N is A, T, C, or G) known to be required for Cas9 activity. Many Cas9 orthologs from a variety of species have been identified herein, and these proteins share only a few identical amino acids. All identified Cas9 orthologs have the same domain architecture as the central HNH endonuclease domain and the separate RuvC / RNase H domain. Cas9 proteins share four key motifs with conserved structures; motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif. In contrast, Cas12a proteins from different species may have different PAM sequence requirements compared to the canonical PAM of LbCas12a from TTTV.

[0198] The site-directed modification polypeptide can also be a chimeric and modified CRISPR / Cas nuclease. For example, it can be a modified Cas9 "base editor". Base editing enables a target DNA base to be directly and irreversibly converted to another base in a programmable manner without the need for DNA cutting or donor DNA molecules. For example, Komor et al. (2016, Nature [Nature], 533: 420-424) teach Cas9-cytidine deaminase fusion, in which Cas9 has also been engineered to be inactive and does not induce double-stranded DNA breaks. In addition, Gaudelli et al. (2017, Nature [Nature], doi: 10.1038 / nature24644) teach catalytically impaired Cas9 fused to tRNA adenosine deaminase, which can mediate the conversion of A / T to G / C in the target DNA sequence. Another class of engineered Cas9 nucleases that can serve as site-directed modifying polypeptides in the methods and compositions of the present invention are variants that can recognize a broad range of PAM sequences, including NG, GAA, and GAT (Hu et al., 2018, Nature, doi: 10.1038 / nature26155).

[0199] Example

[0200] In one embodiment, we provide a fusion protein comprising a heterologous domain, a first linker sequence, and a V-type CRISPR-Cas enzyme in the direction of N-terminus to C-terminus, wherein the first linker sequence comprises a repeated GGGGS sequence. On the one hand, the heterologous domain is a deaminase, a polymerase, a nuclease, a relaxase, an alkyltransferase, a methyltransferase, adenosine deaminase, a cytidine deaminase, an oxidase, a thymine alkyltransferase, adenine oxidase, adenosine methyltransferase, a glycosylase, or a nuclear localization signal. On the other hand, the heterologous domain is a deaminase domain. On the other hand, the deaminase domain is a cytidine deaminase. On the other hand, the cytidine deaminase domain is an activation-induced cytidine deaminase ("AID"). On the other hand, the cytidine deaminase domain is an apolipoprotein B mRNA-editing complex ("APOBEC") domain. On the other hand, the APOBEC domain is an APOBEC1 family deaminase. In yet another aspect, the APOBEC domain comprises a sequence at least 70% identical to SEQ ID NO: 1. In another aspect, the deaminase domain is an adenine deaminase. In yet another aspect, the adenine deaminase is a TadA domain comprising a sequence at least 70% identical to SEQ ID NO: 92.

[0201] In one aspect, the V-type CRISPR-Cas enzyme is a VA-type ("Cas12a") enzyme. In another aspect, the Cas12a domain is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. In yet another aspect, the Cas12a domain is catalytically inactive and is selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 22.

[0202] In one aspect, the first linker sequence comprises GGGGS repeated at least three times. In another aspect, the first linker sequence comprises GGGGS repeated at least six times.

[0203] On the one hand, the fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 13 and 44. On the other hand, the fusion protein further comprises a uracil DNA glycosylase inhibitor ("UGI") domain. On the other hand, the UGI domain comprises SEQ ID NO: 8. On the other hand, the UGI domain is connected to the Cas12a enzyme via a second linker comprising the sequence SGGS. On the other hand, the fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. In another aspect, the fusion protein produces on-target edits at an increased frequency and off-target edits at a decreased frequency when contacted with DNA compared to a fusion protein having a first linker sequence in addition to a repeated GGGGS sequence.

[0204] In another embodiment, we provide a method for editing plant genomic DNA, the method comprising contacting the plant genomic DNA with: (a) the fusion protein of the above aspects, and the fusion protein optionally comprises a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target edits compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence.

[0205] In another embodiment, we provide a method for editing plant genomic DNA with reduced off-target editing, the method comprising contacting the plant genomic DNA with: (a) the fusion protein of the above aspect, and the fusion protein optionally comprising a UGI domain; and (b) a guide RNA ("gRNA") that targets the fusion protein of step (a) to a target DNA sequence of the plant genomic DNA; wherein the edited plant genomic DNA comprises reduced off-target editing compared to plant genomic DNA edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the fusion protein comprises SEQ ID NO: 24.

[0206] In another embodiment, we provide a method for obtaining an edited plant population with reduced off-target editing, the method comprising: (a) obtaining a population of plant cells comprising genomic DNA to be edited; (b) obtaining a nucleotide sequence encoding the fusion protein of the above aspects, and optionally a UGI domain; (c) transforming the population of plant cells with the nucleotide sequence of step (b), thereby expressing the fusion protein encoded by the nucleic acid sequence within the population of plant cells; (d) growing the population of transformed plant cells into plants, wherein at least one plant is edited; and (e) selecting at least one edited plant from the products of step (d), thereby obtaining an edited plant population; wherein the edited plant population comprises reduced off-target editing compared to plants edited by a fusion protein having a first linker other than a repeated GGGGS sequence. In one aspect, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89. In some embodiments, codon-optimized polynucleotides are provided that encode fusion proteins comprising one or more DNA binding domains and one or more DNA modification domains connected by an improved linker sequence.

[0207] Examples

[0208] The following examples provide a number of illustrative embodiments. Based on this disclosure and the general level of skill in the art, one of ordinary skill will appreciate that the following examples are intended to be illustrative only and that many changes, modifications, and variations may be employed without departing from the scope of the subject matter of this disclosure.

[0209] Example 1. Vector construction and guide RNA expression of dLbCas12a-BE

[0210] We fused a catalytically inactive Lachnospiraceae Cas12a containing the D832A / E925A / D1148A mutations (hereafter "dLbCas12a," formerly known as dLbCpf1), rat cytidine deaminase (APOBEC1), and uracil DNA glycosylase inhibitor (UGI) into a single protein via an amino acid linker to exploit the beneficial properties of base editing in plants. The fusion construct was codon-optimized for maize (Zea mays) and commercially synthesized (GenScript, Nanjing, China) and cloned under the promoter of the sugarcane ubiquitin-4 (SoUbi4) gene to constitutively produce dLbCas12a-BE.

[0211] In the dLbCas12a-BE of construct 24524, the nuclear localization signal (SV40-NLS) is in front of APOBEC1 connected to dLbCas12a via an XTEN protein linker, followed by SV40-NLS connected to UGI via an SGGS linker. SV40-NLS is also incorporated into the C-terminus of UGI via an SGGS linker to improve the targeting of the fusion protein to the nucleus. The synthetic sequence of dLbCas12a-BE made using maize optimized codons is listed in SEQ ID NO: 18.

[0212] In the dLbCas12a-BE of construct 24904, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a by a 30 amino acid linker GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 11) with six GGGGS amino acid repeats (referred to as (G4S) x6), followed by the SV40NLS connected to UGI via an SGGS linker. SV40-NLS is also incorporated into the C-terminus of UGI via an SGGS linker to improve the targeting of the fusion protein to the nucleus. The synthetic sequence of dLbCas12a-BE made using maize optimized codons is listed in SEQ ID NO: 23.

[0213] In the dLbCas12a-BE of construct 25057, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a via an XTEN protein linker, followed by the SV40-NLS connected to UGI via an 18 amino acid linker GGSTG GGSGG GSGGG SSG (SEQ ID NO: 12) (referred to as SX). The SV40-NLS is also incorporated into the C-terminus of UGI via a 15 amino acid linker GGGGS GGGGS (referred to as (G4S) x3) to improve the targeting of dLbCas12a-BE to the nucleus. The dLbCas12a-BE synthetic sequence made using maize optimized codons is listed in SEQ ID NO: 14.

[0214] In the dLbCas12a-BE of construct 25058, the SV40-NLS is in front of APOBEC1 connected to dLbCas12a via a 30 amino acid linker ((G4S)x6), followed by the SV40-NLS connected to UGI via an SX linker. SV40-NLS is also incorporated into the C-terminus of UGI via (G4S)x3 to improve dLbCas12a-BE targeting to the nucleus. The dLbCas12a-BE synthetic sequence made using maize optimized codons is listed in SEQ ID NO: 16.

[0215] In the dLbCas12a-BE construct, CRISPR / Cas12a guide RNA transcripts are expressed under the control of the SoUbi4 promoter, which targets the corn Waxy1 exon 4 region, following the PAM sequence in exon 4 to change C9, C10, or C22 to T. It also includes a direct repeat of the LbCrRNA as a scaffold. The synthetic sequence of the guide RNA is listed in SEQ ID NO: 26.

[0216] In construct 24784, a nuclear localization signal (xSV40NLS-06) precedes a cytidine deaminase (xAPOBEC1-01) linked to a maize-optimized Cas9 gene (cCas9BE-02) via xXTEN-02, followed by a nuclear localization signal (xSV40NLS-04) linked to a uracil DNA glycosylase inhibitor xUGI-02 via an xSGGS linker-02. The uracil DNA glycosylase inhibitor xUGI-02 is linked to a nuclear localization signal xSV40NLS-07 via an xSGGS linker-02. The fusion protein is driven by the sugarcane ubiquitin-4 promoter (prSoUbi4-02) and subsequently by the NOS terminator (tNOS-05-01). The Cas9 protein is a nickase Cas9 mutant in which D10A is fused to rat APOBEC1 and a uracil DNA glycosylase inhibitor (UGI). A nuclear localization signal was also incorporated into the C-terminus of Cas9 to improve its targeting to the nucleus. The synthetic sequence of cCas9BE-02 is listed in SEQ ID NO: 20.

[0217] Example 2. Agrobacterium-mediated transformation of maize embryos

[0218] To generate potential events for editing in maize Wx1, the elite maize transformation variety NP2222 was selected for all experiments described (WO 16106121, incorporated herein by reference).

[0219] Maize variety NP2222 was used for maize transformation. Maize ears were harvested from GH when the immature embryos were about 1.2 mm, and then sterilized with 20% Clorox solution for 20 minutes and rinsed three times with sterile water.

[0220] The Agrobacterium tumefaciens strain LBA4404 17740RecA carrying the vector was electroporated - Streak on YP medium containing Gent (25 μg / ml) and Spec (100 μg / ml) antibiotics and grow at 28°C for 2 days. Before transformation, select a single colony and streak it onto a fresh YP plate and grow at 28°C for 1 day. Resuspend the Agrobacterium using the inoculation medium. 660 Adjust to 0.25.

[0221] We removed the endosperm and then separated and collected the immature embryos with a sterile scalpel and injected them into the Agrobacterium suspension for two to three minutes. The infected immature embryos were transferred to co-cultivation medium at 22°C for two to four days.

[0222] After the co-cultivation period, the embryos are transferred to a medium containing the selection agent and maintained in the dark at 28°C for four weeks. Resistant embryonic callus is transferred to regeneration medium and cultured at 28°C with a 16 / 8 photoperiod. Approximately three weeks later, the regenerated plantlets are transferred to a growth container containing rooting medium under the same culture temperature and light conditions.

[0223] Example 3. Analysis of edited bases in target regions

[0224] We used Phire Plant Direct PCR Master Mix (Thermo Fisher, F160L) to amplify a DNA fragment of approximately 410 bp containing the target region directly from maize leaf samples. No DNA purification was required before PCR. The amplified DNA fragment was subjected to Sanger DNA sequencing to analyze mutations at the target site.

[0225] DNA extraction and PCR amplification were performed according to the manufacturer's recommendations. A young leaf (e.g., a punch approximately 2 mm in diameter) was placed in 30 μL of dilution buffer. The leaf sample was crushed using a 100 μL pipette tip by briefly pressing the tube wall and adding 20 μL of dilution buffer. After crushing the leaf, the solution appeared green. The plant material was spun down in a centrifuge, and 1 μL of the supernatant was used as the template for a 20 μL PCR reaction.

[0226] The PCR system consists of the following:

[0227]

[0228] PCR primers for ZmWaxy1:

[0229] Forward primer: 5'-AGATGGGAGACGGGTACGAGACGG-3' (SEQ ID NO: 29)

[0230] Reverse primer: 5'-GTATGGGTTGTTGTTGAGGCTCAGG-3' (SEQ ID NO: 30)

[0231] DNA sequencing primer: 5'-GACCACCCACTGTTCCTGGAGAGGG-3' (SEQ ID NO: 31)

[0232] PCR conditions:

[0233] 98°C for 5 minutes;

[0234] 35 cycles: 98°C for 5 seconds, followed by 60°C for 5 seconds;

[0235] 72°C for 20 seconds;

[0236] 72°C for 1 minute; and

[0237] Keep at 4 °C until ready for analysis.

[0238] Sequencing:

[0239] PCR products were separated by agarose gel electrophoresis and purified with specific primers before Sanger DNA sequencing. For heterozygous mutations, double peaks were observed at the target nucleotide position, while a single peak different from the control was considered a homozygous mutation. Transgenic events with constructs 24524, 24904, and 24784 were used to amplify the ZmWxy1 exon 4 region for sequencing to evaluate base editing.

[0240] Table 1. CRISPR / Cas cytidine base editors (“CBE”) APOBECs containing an XTEN linker between a cytidine deaminase and a nickase Cas9 (“nCas9-CBE”).

[0241]

[0242] The edited nucleotides are shaded in gray. As shown above, this version of the Cas12a base editor, containing an XTEN linker between the APOBEC domain and the site-directed nuclease, most efficiently edits cysteine ​​to thiamine at positions 5 and 6. However, there are instances of guanine to adenine editing at positions -2, 7, and 49. The position is determined by the number of nucleotides from the start of the PAM site.

[0243] Table 2. CRISPR / Cas cytidine base editors containing an XTEN linker between APOBEC deaminases and dLbCas12a

[0244]

[0245] The edited nucleotides are shaded in gray. In this version, the Cas12a base editor, containing an XTEN linker between the APOBEC domain and the inactivated site-directed nuclease, edits cysteine ​​to thiamine at positions 9, 10, and 22, and edits guanine to adenine at positions 39, 44, 52, and particularly 53. The editing of guanine to adenine indicates that the editing occurred on the complement chain.

[0246] Table 3. CRISPR / Cas cytidine base editors containing long linkers between deaminases and dLbCas12a

[0247]

[0248]

[0249] The edited nucleotides are shaded in gray. In this version, a Cas12a base editor containing a long linker containing (G4S)6 between the APOBEC domain and the inactivated site-directed nuclease edits cysteine ​​to thiamine at positions 9 and 10, and edits guanine to adenine at positions 19 and 53. The editing of guanine to adenine indicates that the editing occurred on the complement strand.

[0250] Example 4. Measuring editing efficiency.

[0251] Table 4. Base editing efficiency of maize Wxy1 by the dLbCas12a-CBE system.

[0252]

[0253] Table 4 shows how the base editing efficiency of Cas12a with a long linker is comparable to that of Cas9. Without optimization, Cas12aBE has a poor editing efficiency of approximately 5%, far lower than the editing efficiency of Cas9 (87%). However, by adding a long linker to operably connect the deaminase to the catalytically inactive Cas12a, the editing efficiency increased 12-fold.

[0254] Table 5. Editing efficiency of SBEIIb by LbCas12a with long linkers.

[0255]

[0256] Table 5 shows a direct comparison of the editing efficiency of LbCas12a base editors when operably linked to XTEN linkers or long linkers. When the deaminase is operably linked to the site-directed nuclease through a long linker (such as (G4S)6), the editing efficiency of difficult targets is increased by nearly 5 times.

[0257] Table 6. Editing efficiency of Waxy1 by LbCas12a with long linkers.

[0258]

[0259] Table 7. Multiplex editing of SBEIIb, Waxy1, and Glossy2 by LbCas12a with long linkers.

[0260]

[0261] High editing efficiencies were achieved using multiple simultaneous editing of several guide RNA molecules within the same construct ("multiplexing" or "multiplex editing") and a long linker between the nuclear localization signal and the active Cas12a. Even challenging targets, such as SBEIIb, achieved acceptable editing efficiencies when used as part of a multiplex editing experimental design.

[0262] Example 5. Improved editing in soybean.

[0263] The combination of long linkers and Cas12a also significantly improved soybean editing, with standard Cas12a and long linker-Cas12a improving GmFAD2 editing by nearly 7-fold.

[0264] Table 8. GmFAD2 editing

[0265]

[0266] Example 6. Long linkers improve Mb2Cas12a editing in maize.

[0267] Long linkers also improve the editing efficiency of alternative Cas12 enzymes, such as Mb2Cas12a.

[0268] Table 9. Editing by Mb2Cas12a with long linkers.

[0269]

[0270] Without the long linker, Mb2Cas12a did not edit the target sequence. However, with the long linker, the editing efficiency was significantly improved.

[0271] Example 7. Other heterologous domains are operably linked to Cas12a via long linkers.

[0272] It is also within the scope of the present invention that a heterologous domain (except only APOBEC deaminase) is connected to Cas12a by a long linker. Such heterologous domains include, but are not limited to, deaminases, polymerases, nucleases, relaxases, alkyltransferases, methyltransferases, adenosine deaminase, cytidine deaminase, oxidases, thymine alkyltransferases, adenine oxidase, adenosine methyltransferases, glycosylases or nuclear localization signals.

[0273] We operably linked adenine deaminase to Cas12a to create the Cas12a adenine base editor ("Cas12a-ABE"). We fused catalytically inactive LbCas12a (containing D832A, E925A, and D1148A mutations) to wild-type adenine deaminase from Escherichia coli (engineered "TadA" containing W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N amino acid substitutions) via an amino acid linker. The fusion construct was codon-optimized for maize (Zea mays) and commercially synthesized (GenScript, Nanjing, China) and cloned under the promoter of the Sugarcane Ubiquitin-4 (SoUbi4) gene to constitutively produce dLbCal2a-ABE.

[0274] In the dLbCas12a-ABE of construct 25459, a 189 bp potato intron was inserted into the TadA coding sequence, which was preceded by a TadA variant connected by an XTEN protein linker to produce a TadA dimer. This was fused to dLbCas12a, and the SV40-NLS was incorporated into the C-terminus of dLbCas12a via a GS linker to improve the targeting of the fusion protein to the nucleus. The dLbCas12a-ABE synthetic sequence made using maize optimized codons is listed in SEQ ID NO: 79.

[0275] In the dLbCas12a-ABE of construct 25504, the potato intron of 189bp is inserted into the TadA coding sequence, which is preceded by a TadA variant to produce a TadA dimer. It is connected to dLbCas12a by a 30 amino acid linker (G4S) x6 protein linker, and SV40-NLS is incorporated into the C-terminus of dLbCas12a by a GS linker to improve the targeting of the fusion protein to the nucleus. The dLbCas12a-ABE synthetic sequence manufactured using corn optimized codons is listed in SEQ ID NO:81.

[0276] In the dLbCas12a-ABE construct, CRISPR / Cas12a guide RNA transcripts are expressed under the control of the SoUbi4 promoter, which targets the maize Waxy1 gene. It also includes a direct repeat of the LbCrRNA as a scaffold. The synthetic sequence of the guide RNA is listed in SEQ ID NO: 74.

[0277] In experiments with construct 25459, in which adenine deaminase was linked to dLbCas12a via an XTEN linker, no detectable editing occurred when used in corn plants. In experiments with construct 25504, in which adenine deaminase was linked to dLbCas12a via a (G4S)*6 long linker, an editing efficiency of 7% was achieved, approximately half that of the Cas9ABE control (construct 24785). See Table 10.

[0278] Table 10.dLbCas12aABE

[0279]

[0280] We believe this represents the first time that Cas12aABE has been demonstrated to be effective in plants. It is believed that the use of a long linker to operably link adenine deaminase to Cas12a is the reason for the success of this technology.

[0281] Example 8. Dual base editor in corn

[0282] Dual base editors (fused to the cytidine deaminase domain and adenine deaminase domain of the Cas enzyme). In this concept, targeted saturation mutagenesis of crop genes can be applied to generate genetic variants with improved agronomic performance, for example, C:G>T:A and A:T>G:C substitutions in the same target region. We multiplexed four guide RNAs: one targeting the ZmWaxy1 gene, and three different guide RNAs targeting the ZmADH gene.

[0283] Table 11. Dual CBE-ABE Cas12a editing frequency in maize.

[0284]

[0285] Table 12. Editing in maize by dual CBE-ABE Cas12a.

[0286]

[0287] In total, the dLbCas12a-based CBE-ABE resulted in 1% C to T and A to G mutations. Adding an intron increases vector stability but may reduce enzyme activity due to inefficient splicing. This is believed to be the first example of dual CBE-ABE editing in plants using Cas12a.

[0288] Summary Table

[0289]

[0290]

[0291]

[0292] In the table above, most Cas12aBE constructs follow the pattern of heterologous enzyme domain-linker-Cas enzyme. The exceptions to this pattern are: 25702 [TadA dimer-linker-PmCDA-linker-Cas enzyme], 25701 [PmCDA-linker-TadA dimer-linker-Cas enzyme], and 25658 [TadA dimer-linker-Cas enzyme-PmCDA]. Additional nuclear localization sequences, uracil glycosylase inhibitors, and other components may be present but are not shown in this table. Such details appear in the sequences provided in the accompanying sequence table.

[0293] These examples and embodiments provided herein are non-limiting illustrations of the claims and should not be construed as the only available examples. Additional variations may occur to those skilled in the art. Sequence Listing <110> Syngenta Li, Jiang Xu, Jianping <120> Methods and compositions for DNA base editing <130> Agent Case No. 81945 <160> 92 <170> PatentIn version 3.5 <210> 1 <211> 229 <212> PRT <213> Brown rat (Rattus norvegicus) <400> 1 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val 50 55 60 Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser 145 150 155 160 Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp 210 215 220 Ala Thr Gly Leu Lys 225 <210> 2 <211> 687 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 2 atgtccagcg agaccggccc cgtggcggtg gaccccaccc tgcgcaggcg catcgagccg cacgagttcg aggtgttctt cgaccccagg gagctccgca aggagacctg cctcctgtac gagatcaact ggggcggcag gcactccatc tggaggcaca cgagccagaa caccaacaag cacgtcgagg tgaacttcat cgagaagttc accacggaga ggtacttctg cccgaacacg cgctgctcca tcacgtggtt cctctcgtgg agcccatgcg gcgagtgctc cagggcgatc acggagttcc tcagccgcta cccgcacgtg accctgttca tctacatcgc taggctctac caccacgcgg accccaggaa caggcagggc ctcagggacc tgatctccag cggcgtcacg atccagatca tgaccgagca ggagtccggc tactgctgga ggaacttcgt gaactactcc ccgagcaacg agccccactg gccccgctac ccgcaccctc gggtccgcct ctacgtgctc 540 gagctgtact gcatcatcct cggcctgccg ccctgcctca acatcctgag gcgcaagcag 600 ccccagctga cgttcttcac catcgccctg cagagctgcc actaccagag gctcccgccc 660 cacatcctgt gggcgaccgg gctcaag 687 <210> 3 <211> 1251 <212> PRT <213> Moraxella bovis <400> 3 Met Leu Phe Gln Asp Phe Thr His Leu Tyr Pro Leu Ser Lys Thr Val 1 5 10 15 Arg Phe Glu Leu Lys Pro Ile Gly Arg Thr Leu Glu His Ile His Ala 20 25 30 Lys Asn Phe Leu Ser Gln Asp Glu Thr Met Ala Asp Met Tyr Gln Lys 35 40 45 Val Lys Val Ile Leu Asp Asp Tyr His Arg Asp Phe Ile Ala Asp Met 50 55 60 Met Gly Glu Val Lys Thr Lys Leu Ala Glu Phe Tyr Asp Val Tyr 65 70 75 80 Leu Lys Phe Arg Lys Asn Pro Lys Asp Gly Leu Gln Lys Gln Leu 85 90 95 Lys Asp Leu Gln Ala Val Leu Arg Lys Glu Ser Val Lys Pro Ile Gly 100 105 110 Ser Gly Gly Lys Tyr Lys Thr Gly Tyr Asp Arg Leu Phe Gly Ala Lys 115 120 125 Leu Phe Lys Asp Gly Lys Glu Leu Gly Asp Leu Ala Lys Phe Val Ile 130 135 140 Ala Gln Glu Gly Glu Ser Ser Pro Lys Leu Ala His Leu Ala His Phe 145 150 155 160 Glu Lys Phe Ser Thr Tyr Phe Thr Gly Phe His Asp Asn Arg Lys Asn 165 170 175 Met Tyr Ser Asp Glu Asp Lys His Thr Ala Ile Ala Tyr Arg Leu Ile 180 185 190 His Glu Asn Leu Pro Arg Phe Ile Asp Asn Leu Gln Ile Leu Thr Thr 195 200 205 Ile Lys Gln Lys His Ser Ala Leu Tyr Asp Gln Ile Ile Asn Glu Leu 210 215 220 Thr Ala Ser Gly Leu Asp Val Ser Leu Ala Ser His Leu Asp Gly Tyr 225 230 235 240 His Lys Leu Leu Thr Gln Glu Gly Ile Thr Ala Tyr Asn Arg Ile Ile 245 250 255 Gly Glu Val Asn Gly Tyr Thr Asn Lys His Asn Gln Ile Cys His Lys 260 265 270 Ser Glu Arg Ile Ala Lys Leu Arg Pro Leu His Lys Gln Ile Leu Ser 275 280 285 Asp Gly Met Gly Val Ser Phe Leu Pro Ser Lys Phe Ala Asp Asp Ser 290 295 300 Glu Met Cys Gln Ala Val Asn Glu Phe Tyr Arg His Tyr Thr Asp Val 305 310 315 320 Phe Ala Lys Val Gln Ser Leu Phe Asp Gly Phe Asp Asp His Gln Lys 325 330 335 Asp Gly Ile Tyr Val Glu His Lys Asn Leu Asn Glu Leu Ser Lys Gln 340 345 350 Ala Phe Gly Asp Phe Ala Leu Leu Gly Arg Val Leu Asp Gly Tyr Tyr 355 360 365 Val Asp Val Val Asn Pro Glu Phe Asn Glu Arg Phe Ala Lys Ala Lys 370 375 380 Thr Asp Asn Ala Lys Ala Lys Leu Thr Lys Glu Lys Asp Lys Phe Ile 385 390 395 400 Lys Gly Val His Ser Leu Ala Ser Leu Glu Gln Ala Ile Glu His His 405 410 415 Thr Ala Arg His Asp Asp Glu Ser Val Gln Ala Gly Lys Leu Gly Gln 420 425 430 Tyr Phe Lys His Gly Leu Ala Gly Val Asp Asn Pro Ile Gln Lys Ile 435 440 445 His Asn Asn His Ser Thr Ile Lys Gly Phe Leu Glu Arg Glu Arg Pro 450 455 460 Ala Gly Glu Arg Ala Leu Pro Lys Ile Lys Ser Gly Lys Asn Pro Glu 465 470 475 480 Met Thr Gln Leu Arg Gln Leu Lys Glu Leu Leu Asp Asn Ala Leu Asn 485 490 495 Val Ala His Phe Ala Lys Leu Leu Thr Thr Lys Thr Thr Leu Asp Asn 500 505 510 Gln Asp Gly Asn Phe Tyr Gly Glu Phe Gly Val Leu Tyr Asp Glu Leu 515 520 525 Ala Lys Ile Pro Thr Leu Tyr Asn Lys Val Arg Asp Tyr Leu Ser Gln 530 535 540 Lys Pro Phe Ser Thr Glu Lys Tyr Lys Leu Asn Phe Gly Asn Pro Thr 545 550 555 560 Leu Leu Asn Gly Trp Asp Leu Asn Lys Glu Lys Asp Asn Phe Gly Val 565 570 575 Ile Leu Gln Lys Asp Gly Cys Tyr Tyr Leu Ala Leu Leu Asp Lys Ala 580 585 590 His Lys Lys Val Phe Asp Asn Ala Pro Asn Thr Gly Lys Asn Val Tyr 595 600 605 Gln Lys Met Val Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro 610 615 620 Lys Val Phe Phe Ala Lys Ser Asn Leu Asp Tyr Tyr Asn Pro Ser Ala 625 630 635 640 Glu Leu Leu Asp Lys Tyr Ala Lys Gly Thr His Lys Lys Gly Asp Asn 645 650 655 Phe Asn Leu Lys Asp Cys His Ala Leu Ile Asp Phe Phe Lys Ala Gly 660 665 670 Ile Asn Lys His Pro Glu Trp Gln His Phe Gly Phe Lys Phe Ser Pro 675 680 685 Thr Ser Ser Tyr Arg Asp Leu Ser Asp Phe Tyr Arg Glu Val Glu Pro 690 695 700 Gln Gly Tyr Gln Val Lys Phe Val Asp Ile Asn Ala Asp Tyr Ile Asp 705 710 715 720 Glu Leu Val Glu Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys 725 730 735 Asp Phe Ser Pro Lys Ala His Gly Lys Pro Asn Leu His Thr Leu Tyr 740 745 750 Phe Lys Ala Leu Phe Ser Glu Asp Asn Leu Ala Asp Pro Ile Tyr Lys 755 760 765 Leu Asn Gly Glu Ala Gln Ile Phe Tyr Arg Lys Ala Ser Leu Asp Met 770 775 780 Asn Glu Thr Thr Ile His Arg Ala Gly Glu Val Leu Glu Asn Lys Asn 785 790 795 800 Pro Asp Asn Pro Lys Lys Arg Gln Phe Val Tyr Asp Ile Ile Lys Asp 805 810 815 Lys Arg Tyr Thr Gln Asp Lys Phe Met Leu His Val Pro Ile Thr Met 820 825 830 Asn Phe Gly Val Gln Gly Met Thr Ile Lys Glu Phe Asn Lys Lys Val 835 840 845 Asn Gln Ser Ile Gln Gln Tyr Asp Glu Val Asn Val Ile Gly Ile Asp 850 855 860 Arg Gly Glu Arg His Leu Leu Tyr Leu Thr Val Ile Asn Ser Lys Gly 865 870 875 880 Glu Ile Leu Glu Gln Arg Ser Leu Asn Asp Ile Thr Thr Ala Ser Ala 885 890 895 Asn Gly Thr Gln Val Thr Thr Pro Tyr His Lys Ile Leu Asp Lys Arg 900 905 910 Glu Ile Glu Arg Leu Asn Ala Arg Val Gly Trp Gly Glu Ile Glu Thr 915 920 925 Ile Lys Glu Leu Lys Ser Gly Tyr Leu Ser His Val Val His Gln Ile 930 935 940 Asn Gln Leu Met Leu Lys Tyr Asn Ala Ile Val Val Leu Glu Asp Leu 945 950 955 960 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Ile Tyr 965 970 975 Gln Asn Phe Glu Asn Ala Leu Ile Lys Lys Leu Asn His Leu Val Leu 980 985 990 Lys Asp Lys Ala Asp Asp Glu Ile Gly Ser Tyr Lys Asn Ala Leu Gln 995 1000 1005 Leu Thr Asn Asn Phe Thr Asp Leu Lys Ser Ile Gly Lys Gln Thr 1010 1015 1020 Gly Phe Leu Phe Tyr Val Pro Ala Trp Asn Thr Ser Lys Ile Asp 1025 1030 1035 Pro Glu Thr Gly Phe Val Asp Leu Leu Lys Pro Arg Tyr Glu Asn 1040 1045 1050 Ile Ala Gln Ser Gln Ala Phe Phe Gly Lys Phe Asp Lys Ile Cys 1055 1060 1065 Tyr Asn Thr Asp Lys Gly Tyr Phe Glu Phe His Ile Asp Tyr Ala 1070 1075 1080 Lys Phe Thr Asp Lys Ala Lys Asn Ser Arg Gln Lys Trp Ala Ile 1085 1090 1095 How Is Gly Asp Replaced With Tyr Val Tyr Asp With Thr Ala Asn 1100 1105 1110 Gln Asn Lys Gly Ala Ala Lys Gly Ile Asn Val Asn Asp Glu Leu 1115 1120 1125 Lys Ser Leu Phe Ala Arg Tyr His Ile Asn Asp Lys Gln Pro Asn 1130 1135 1140 Leu Val Met Asp With Cys Gln Asn Asp Lys Glu Phe His Lys 1145 1150 1155 Ser Lew Met Cys Lew Lew Lys Thr Lew Lew Ala Lew Arg Tyr Ser 1160 1165 1170 Asn Ala Ser Ser Asp Glu Asp Phe Ile Leu Ser Pro Val Ala Asn 1175 1180 1185 Asp Glu Gly Val Phe Phe Asn Ser Ala Leu Ala Asp Asp Thr Gln 1190 1195 1200 Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys 1205 1210 1215 Gly Leu Trp Leu Leu Asn Glu Leu Lys Asn Ser Asp Asp Leu Asn 1220 1225 1230 Lys Val Lys Leu Ala Ile Asp Asn Gln Thr Trp Leu Asn Phe Ala 1235 1240 1245 Gln Asn Arg 1250 <210> 4 <211> 3753 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 4 gctctgtttc aagattttac acatctgtac ccgctgagta aaacagtgcg gttcgagctg 60 aaacccatag gaaggaccct cgagcacatc cacgcgaaga attttctgag ccaggatgaa 120 actatggctg atatgtatca aaaagttaag gtcattttgg acgactatca tcgcgatttt 180 attgccgaca tgatgggaga ggtgaaactc acgaagcttg ctgaatttta cgacgtctat 240 ctgaagttca ggaaaaatcc taggacgat gggctgcaaa aacagcttaa agaccttcaa 300 gctgtccttc ggaaggaatc ggtgaagcct atagggtcag gtgggaagta caaaacaggc 360 tacgatagac tctttggggc aaaactcttc aaagatggaa aagagttggg tgacctcgca 420 aaattcgtta tagcccaaga aggtgagtct tctccgaagc tggctcatct tgctcatttt 480 gagaagttca gcacgtattt tactggattt cacgataatc ggaagaatat gtactcggat 540 gaagacaagc atactgcaat agcgtacagg ctcatccatg agaatttgcc gagattcatc 600 gacaatctgc aaatcttgac aacaatcaaa caaaagcata gcgccctcta tgatcagata 660 atcaacgagc tcacggcctc cgggctcgac gtctccttgg cttctcatct tgacgggtat 720 caaagctcc ttacacaaga ggggatcacg gcatacaaca ggatcatagg agaggtgaat 780 ggatatacaa ataagcataa ccagatatgc cacaagagcg agcgcatagc gaaacttaga 840 cccttgcaca agcaaatcct ttctgacgga atgggagtgt cattccttcc gtctaagttc 900 gcggatgata gtgagatgtg ccaagcggtc aacgaatttt atcgccatta tactgacgtg 960 ttcgcaaagg tgcaaagtct ctttgacgga tttgatgatc accagaaaga cgggatctat gttgaacaca aaaaccttaa tgaactgagc aaacaggcgt tcggcgactt tgctttgctg gggagggtcc ttgatggata ctacgtggac gttgtcaatc cggagttcaa tgagcggttc 1140 gcaaaggcca agactgacaa tgcgaaagcc aagcttacaa aagaaagga caaattcatt aaggagtcc actcactggc ttccctcgaa caagcaatag aacaccatac agctagacac 1320. gcgatgaga gtgttcaagc cggaaaactt ggccagtact tcaaacacgg tttggcgggg gttgacaacc cgattcagaa aattcacaat aaccattcga cgattaaagg gtttctggaa agggaaggc ctgctgggga acggggcgctc ccgaagatca agtcaggaa aaacccagaa atgacacagc tcaggcagct gaaggaactt ttggacaacg cattgaatgt ggcgcacttc gctaagctgc tgacaactaa aacaaccttg gacaaccagg atggaaattt ttacggggag tttggggtgc tttacgacga gctggctaaa attccaactc tctacaataa ggttagagat tatctctctc aaaagccctt ttctaccgaa aagtataagc tcaacttcgg caatccgacc 1680. cttctcaatg ggtgggacct gaacaaagag aaagataact ttggggttat acttcagaag 1740 gatggatgct attacttggc gcttcttgat aaggctcata aaaaagtttt cgacaacgcc 1800 cctaacactg gtaagaacgt ctaccaaaag atggtctaca aactgttgcc cggccccaac 1860 aaaatgcttc ctaaagtgtt tttcgcaaaa tcgaatctcg actattataa tccatctgcc 1920 gagctccttg acaaatatgc taaggggacc cataaaaagg gtgataattt caacctgaag 1980 gactgccacg cgcttatcga ctttttcaaa gccgggataa ataagcatcc ggagtggcaa 2040 cattttggtt ttaaattttc gccaacgtcg tcctatcgcg acctttccga tttctatagg 2100 gaagttgaac ctcaggggta ccaggtcaaa tttgttgaca ttaatgcgga ctacattgat 2160 gaattggtgg agcaagggaa gctctacctc tttcaaatat ataacaaaga tttctcgcca 2220 aaagcgcatg gtaaaccgaa tcttcatacc ttgtacttta aagcactttt ttcagaagat 2280 aacttggcgg acccgatcta caagctgaat ggggaagctc agatcttcta caggaaagct 2340 tcgttggaca tgaacgagac taccatacat cgcgcgggag aggtgcttga gaacaaaaat 2400 cccgacaacc cgaaaaagcg gcaattcgtt tacgacatca tcaaagacaa acggtacacg 2460 caggacaaat ttgctcca cgtccccatt accatgaatt ttggagtcca aggcatgacc 2520 attaaggaat tcaacaaaaa ggtcaaccaa agtattcagc atacgatga agtcaatgtc 2580 ataggcatag atcggggaga aaggcatctg ttgtatctta ccgtgattaa ctctaagggt 2640 gaaatactgg agcaacggtc acttaacgat ataaccacgg cgtccgcgaa cggtacacaa 2700 gtgaccactc cctaccacaa aatattggat aaaagggaga tagaacgctt gaatgcccgc 2760 gttggctggg gtgagattga gaccatcaaa gagcttaaat cgggatattt gtctcacgtc 2820 gttcatcaaa ttaaccaact catgcttaag tacaatgcaa tcgttgtgct cgaggacctg 2880 aactttggtt tcaaaagagg gaggttcaag gtggaaaaac aaatttacca gaactttgaa 2940 aacgcgctta tcaagaaatt gaatcacctt gttttgaaag ataaggcaga tgacgaaatc 3000 gggtcgtata aaaattgcact ccagttgaca aataatttca cggatttgaa gtcgatcggc 3060 aagcaaacag ggttcctct tttgtgcca gcgtggaata catcaaaaat tgatccggag 3120 acgggatttg tcgacttgct gaagcctagg tatgagaaca ttgcccaatc tcaggccttt 3180 ttcggcaaat tcgataaaat atgctacaac acagacaaag gttattttga atttcacatt 3240 gattacgcca aatttacaga taaggcgaaa aacagcagac agaaatgggc tatctgttct 3300 catggggaca aacgctatgt ctacgataag acggctaatc aaaataaagg cgccgcaaa 3360 ggtattaatg tgaatgatga gctgaaaagc ttgtttgccc gctaccatat caatgataaa 3420 caaccaaact tggtgatgga catatgccag aacaatgaca aagaattcca caagtcactc 3480 atgtgcctgc ttaaaaccct tttggcgctg cggtatagca atgcatctag cgatgaagac 3540 tttattttga gtcccgtggc caacgacgag ggcgtgtttt ttaattcagc cttggcggac 3600 gatacgcagc cccagaatgc ggacgcaaac ggcgcgtacc acattgcact gaagggactg 3660 tggcttctga acgagctgaa aaatagcgac gacctgaata aagtcaagtt ggccattgac 3720 aatcaaacct ggttgaattt cgctcaaaat aga 3753 <210> 5 <211> 4367 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized fusion protein <400> 5 atgtccagcg agaccggccc cgtggcggtg gaccccaccc tgcgcaggcg catcgagccg 60 cacgagttcg aggtgttctt cgaccccagg gagctccgca aggagacctg cctcctgtac 120 gagatcaact ggggcggcag gcactccatc tggaggcaca cgagccagaa caccaacaag 180 cacgtcgagg tgaacttcat cgagaagttc accacggaga ggtacttctg cccgaacacg 240 cgctgctcca tcacgtggtt cctctcgtgg agcccatgcg gcgagtgctc cagggcgatc 300 acggagttcc tcagccgcta cccgcacgtg accctgttca tctacatcgc taggctctac 360 caccacgcgg accccaggaa caggcagggc ctcagggacc tgatctccag cggcgtcacg 420 atccagatca tgaccgagca ggagtccggc tactgctgga ggaacttcgt gaactactcc 480 ccgagcaacg aggcccactg gccccgctac ccgcacctct gggtccgcct ctacgtgctc 540 gagctgtact gcatcatcct cggcctgccg ccctgcctca acatcctgag gcgcaagcag 600 ccccagctga cgttcttcac catcgccctg cagagctgcc actaccagag gctcccgccc 660 cacatcctgt gggcgaccgg gctcaagggg ggcggggggct caggcgggg cgggagcggc 720 ggcggggggct ctggggcgg cggcagcggc gggggcggca gcgggggcgg cgggtcgatg 780 agcaagctgg agaagttcac gaactgctac tccctcagca agaccctgag gttcaaggcg 840 atcccggtcg gcaagaccca ggagaacatc gaacaagc ggctgctggt ggaggacgag 900 aagagggctg aggactacaa gggcgtgaag aagctcctgg accgctacta cctgtccttc 960 atcaacgacg tgctccacag catcaagctc aagaacctga aacactacat cagcctcttc 1020 aggaagaaga cgcgcaccga gaaggagaac aaggaagctcg agaacctgga gatcaacctg 1080 aggaagga tcgccaaggc gttcaagggc aacgagggct acaagtccct cttcaagaag 1140 gacatcatcg agacgatcct cccggagttc ctggacgaca aggacgagat cgccctggtc 1200 aactccttca acggcttcac cacggcgttc accggcttct tcgacaaccg cgagaacatg 1260 ttcagcgagg aggccaagtc cacgagcatc gcgttcaggt gcatcaacga gaacctcacc 1320 1380 gtgcaggaga tcaggaga gatcctgaac agcgactacg acgtcgagga cttcttcgag 1440 ggcgagttct tcaacttcgt cctcacgcag gaggcatcg acgtgtacaa cgccatcatc 1500 ggtggcttcg tgaccgagtc cggcgagaag atcaagggcc tgaacgagta catcaacctc 1560 tacaaccaga agaccaagca gaagctgccg aagttcaagc ccctgtacaa gcaggtgctc 1620 tccgacagggg agtccctcag cttctacggc gaggctaca cgagcgacga ggaggtcctg 1680 gaggtgttcc 1740 gcacaccct spagaacct gagaagctgt tcagaactt cgacgagtac tccagcgccg gcatctcgt caagaacggc 1800 ccggcgatct ccacgatcag caggacatc ttcggcgagt ggaacgtgat ccgcgacag 1860 tggaacgccg agtacgacga catccaccctc aagagaagg cggtggtcac cgagaagtac 1920 gaggacgaca ggcgcaagtc ctcaagaag atcggctcct tcagcctcga gcagctgcag 1980 gagtacgccg acggcggacct gagcgtggtc gagaagctca aggagatcat catchcagaag 2040 gtcgacgaga tctacaaggt gtacggctcc agcgagaagc tctcgacgc ggactcgtc 2100 ctcgagaagt ccctgaagaa gaacgacgcc gtggtcgcga tcatgaagga cctcctggac 2160 tccgtgaaga gcttcgagaa ttacatcaag gccttcttcg gcgagggcaa ggagacgaac 2220 agggacgagt ccttctacgg cgacttcgtc ctggcctacg acatcctcct gaaggtggac 2280 cacatctacg acgcgatccg caactacgtg acccagaagc cgtacagcaa ggacaagttc 2340 aagctctact tccagaaccc ccagttcatg ggcggctggg acaaggacaa ggagacggac 2400 tacagggcga ccatcctgcg ctacggcagc aagtactacc tcgccatcat ggacaagaag 2460 tacgcgaagt gcctgcagaa gatcgacaag gacgacgtca acggcaacta cgagaagatc 2520 aactacaagc tcctgccggg ccccaacaag atgctcccga aggtgttctt ctccaagaag 2580 tggatggcct actacaaccc cagcgaggac atccagaaga tctacaagaa cggcacgttc 2640 aagaagggcg acatgttcaa cctgaacgac tgccacaagc tcatcgactt cttcaaggac 2700 tccatcagcc gctacccgaa gtggtccaac gcctacgact tcaacttcag cgagaccgag 2760 aagtacaagg acatcgcggg cttctaccgc gaggtcgagg agcagggcta caaggtgtcc 2820 ttcgagtccg ccagcaagaa ggaggtcgac aagctggtgg aggaggcaa gctctacatg 2880 ttccagatct acaaagga cttctccgac aagagccacg gcacgcccaa cctgcacacc 2940 atgtacttca agctcctgtt cgacgagaac aaccacggcc agatcaggct gtccggcggc 3000 gccgagctct tcatgaggag ggcgagcctg aagaaggagg agctggtggt ccaccccgct 3060 aacagcccaa tcgcgaaca gaacccggac aaccccaga agaccacgac cctgtcctac 3120 gacgtgtaca aggacaagg gttcagcgag gaccagtacg agctccacat cccgatcgcg 3180 atcacaagt gccccaagaa catctcaag atcacaccg aggtccgcgt gctcctgaag 3240 cacgacgaca acccctacgt gatcggcatc gctaggggcg agaggaacct cctgtacatc 3300 gtggtcgtgg acggcaaggg cacatcgtg gagcagtact ccctcaacga gatcatcac 3360 aacttcaacg gcatcaggat cavacggac taccacagcc tcctggacaca gaggagag 3420 gagaggttcg aggcccgcca gaacggacc tccatcgaga acatcagga gctgaggcg 3480 ggctacatca gccaggtcgt gcacaagatc tgcgagctcg tcgagaagta cgacgccgtg 3540 atcgccctcg cggacctgaa ctccggcttc aagaacagcc gcgtcaaggt ggagaagcag 3600 gtctaccaga agttcgagaa gatgctcatc gacaagctga actacatggt ggacaagaag 3660 tccaacccct gcgctacggg cggcgcgctg aagggctacc agatcaccaa caagttcgag 3720 agcttcaagt ccatgagcac tcagaacggc ttcatcttct acatcccggc gtggctcacg 3780 tccaagatcg accccagcac cggcttcgtc aacctcctga agacgaagta cacctccatc 3840 gccgacagca agaagttcat ctccagcttc gaccgcatca tgtatgtgcc ggaggaggac 3900 ctgttcgagt tcgccctcga ctacaagaac ttctcccgca cggacgcgga ctacatcaag 3960 aagtggaagc tgtacagcta cggcaaccgc atccgcatct tcaggaaccc caagaagaac 4020 aacgtcttcg actgggagga ggtgtgcctg acctccgcgt acaaggagct cttcaacaag 4080 tacggcatca actaccagca gggcgacatc agggctctcc tgtgcgagca gagcgacaag 4140 gccttctact ccagcttcat ggcgctgatg tccctcatgc tgcagatgag gaactcgatc 4200 accggcagga cggacgtggc cttcctcatc tccccggtga agaacagcga cggcatcttc 4260 tacgactcca ggaactacga ggcccaggag aacgcgatcc tcccaaagaa cgcggacgcc 4320 aacggcgcct acaacatcgc caggaaggtc ctctgggcta tcggcca 4367 <210> 6 <211> 1455 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 6 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val 50 55 60 Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser 145 150 155 160 Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp 210 215 220 Ala Thr Gly Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu 260 265 270 Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu 275 280 285 Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu 290 295 300 Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe 305 310 315 320 Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr 325 330 335 Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu 340 345 350 Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe 355 360 365 Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu 370 375 380 Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val 385 390 395 400 Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn 405 410 415 Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe 420 425 430 Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile 435 440 445 Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile 450 455 460 Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu 465 470 475 480 Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr 485 490 495 Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys 500 505 510 Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys 515 520 525 Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu 530 535 540 Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu 545 550 555 560 Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser 565 570 575 Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser 580 585 590 Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys 595 600 605 Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu 610 615 620 Tyr Asp Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr 625 630 635 640 Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu 645 650 655 Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys 660 665 670 Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr 675 680 685 Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser 690 695 700 Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp 705 710 715 720 Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly 725 730 735 Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala 740,745,750 Tyr Asp With Leu Leu Lys Val Asp His With Tyr Asp Ala With Arg Asn 755,760,765 Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe 770,775,780 Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp 785,790,795,800 Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile 805 810 815 Met Asp Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp 820 825 830 Val Asn Gly Asn Tyr Glu Lys And Asn Tyr Lys Leu Pro Gly Pro 835 840 845 Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr 850 855 860 Tyr Asn Pro Ser Glu Asp With Gln Lys And Tyr Lys Asn Gly Thr Phe 865 870 875 880 Lys Gly Asp Met Phe Asn Leu Asp Cys His Lys Gly Asp 885,890,895 Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr 900 905 910 Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe 915 920 925 Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala 930 935 940 Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met 945 950 955 960 Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro 965 970 975 Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His 980 985 990 Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala 995 1000 1005 Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro 1010 1015 1020 Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu 1025 1030 1035 Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr 1040 1045 1050 Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile 1055 1060 1065 Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp 1070 1075 1080 Asn Pro Tyr Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu 1085 1090 1095 Tyr Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr 1100 1105 1110 Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys 1115 1120 1125 Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe 1130 1135 1140 Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu 1145 1150 1155 Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu Leu 1160 1165 1170 Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Ala Asp Leu Asn Ser 1175 1180 1185 Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln 1190 1195 1200 Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp 1205 1210 1215 Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr 1220 1225 1230 Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Th Thr Gln 1235 1240 1245 Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile 1250 1255 1260 Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr 1265 1270 1275 Serve Ala Asp Serve Lys Lys Phe Ile Serve Phe Asp Arg Ile 1280 1285 1290 Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr 1295 1300 1305 Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys 1310 1315 1320 Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys 1325 1330 1335 Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala 1340 1345 1350 Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly 1355 1360 1365 Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr 1370 1375 1380 Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn 1385 1390 1395 Ser Ile Thr Gly Arg Thr Asp Val Ala Phe Leu Ile Ser Pro Val 1400 1405 1410 Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala 1415 1420 1425 Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala 1430 1435 1440 Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly 1445 1450 1455 <210> 7 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> Codon Optimization <F400> 7 acgaacctgt ccgacatcat cgagaaggag accggcaagc agctcgtgat ccaggagagc 60 atcctcatgc tgccggagga ggtcgaggag gtcatcggca acaagcccga gtccgacatc 120 ctcgtccaca cggcctacga cgagtccacc gacgagaacg tgatgctcct gacctcggac 180 gctcccgagt acaagccatg ggccctggtc atccaggaca gcaacggcga gaacaagatc 240 aagatgctc 249 <210> 8 <211> 83 <212> PRT <213> Bacillus subtilis phage PBSX <400> 8 Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val 1 5 10 15 Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile 20 25 30 Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu 35 40 45 Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr 50 55 60 Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile 65 70 75 80 Lys Met Leu <210> 9 <211> 6882 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized fusions <400> 9 gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 acgagaccgc gacgcaaccg cctctcgccg ctgggcccca caccgctcgg tgccgtagcc 300 tcacgggact ctttctccct cctccccgt tataaattgg cttcatcccc tccttgcctc 360 atccatccaa atcccagtcc ccaatcccat cccttcgtag gagaaattca tcgaagctaa 420 gcgaatcctc gcgatcctct caaggtactg cgagttttcg atccccctct cgacccctcg 480 tatgtttgtg tttgtcgtag cgtttgatta ggtatgcttt ccctgtttgt gttcgtcgta 540 gcgtttgatt aggtatgctt tccctgttcg tgttcatcgt agtgtttgat taggtcgtgt 600 gaggcgatgg cctgctcgcg tccttcgatc tgtagtcgat ttgcgggtcg tggtgtagat 660 ctgcgggctg tgatgaagtt atttggtgtg atctgctcgc ctgattctgc gggttggctc 720 gagtagatat gatggttgga ccggttggtt cgtttaccgc gctagggttg ggctgggatg 780 atgttgcatg cgccgttgcg cgtgatcccg cagcaggact tgcgtttgat tgccagatct 840 cgttacgatt atgtgatttg gtttggactt tttagatctg tagcttctgc ttatgtgcca 900 gatgcgccta ctgctcatat gcctgatgat aatcataaat ggctgtggaa ctaactagtt 960 gattgcggag tcatgtatca gctacaggtg tagggactag ctacaggtgt agggacttgc 1020 gtctaattgt ttggtccttt actcatgttg caattatgca atttagttta gattgtttgt 1080 tccactcatc taggctgtaa aagggacact gcttagattg ctgtttaatc tttttagtag 1140 attatattat attggtaact tattacccct attacatgcc atacgtgact tctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttg attgttgaat catgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac tgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaa gaatcaggtt 1380 cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat atatcactgg 1440 aattaattag ttgatcgttt aatcatatat caagtacata ccatgccaca atttttagtc 1500 acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgtt ctattctgat 1560 tagaccatat atcatgtatt ttttttggt aatggttctc ttattttaaa tgctatatag 1620 ttctggtact tgttagaaag atctgcttca tagtttagtt gcctatccct cgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaa ttctttgtg ttcaacagtc 1740 agttttgtt agattcattg taacttatgg tcgcttactc ttctggtcct caatgcttgc 1800 agggatcccc taatagacc atgccgaaga agaagcgcaa ggtcatgtcc agcgagaccg 1860 gccccgtggc ggtggacccc accctgcgca ggcgcatcga gccgcacgag ttcgaggtgt 1920 tcttcgaccc cagggagctc cgcaaggaga cctgcctcct gtacgagatc aactggggcg 1980 gcaggcactc catctggagg cacacgagcc agaacaccaa caagcacgtc gaggtgaact 2040 tcatcgagaa gttcaccacg gagaggtact tctgcccgaa cacgcgctgc tccatcacgt 2100 ggttcctctc gtggagccca tgcggcgagt gctccagggc gatcacggag ttcctcagcc 2160 gctacccgca cgtgaccctg ttcatctaca tcgctaggct ctaccaccac gcggacccca 2220 ggaaggca gggcctcagg gacctgatct ccagcggcgt cacgatccag atcatgaccg 2280 agcaggagtc cggctactgc tggaggaact tcgtgaacta ctccccgagc aacgaggccc 2340 actggccccg ctacccgcac ctctgggtcc gcctctacgt gctcgagctg tactgcatca 2400 tcctcggcct gccgccctgc ctcaacatcc tgaggcgcaa gcagccccag ctgacgttct 2460 tcaccatcgc cctgcagagc tgccactacc agaggctccc gccccacatc ctgtgggcga 2520 ccgggctcaa ggggggcggg ggctcaggcg ggggcgggag cggcggcggg ggctctgggg 2580 gcggcggcag cggcggggc ggcagcgggg gcggcgggtc gatgagcaag ctggagaagt 2640 tcacgaactg ctactccctc agcaagaccc tgaggttcaa ggcgatcccg gtcggcaaga 2700 cccaggagaa catcgacaac aagcggctgc tggtggagga cgagaagagg gctgaggact 2760 acaagggcgt gaaagctc ctggaccgct actacctgtc cttcatcaac gacgtgctcc 2820 acagcatcaa gctcaagaac ctgaacaact acatcagcct cttcaggaag aagacgcgca 2880 ccgagaagga gaacaggag ctcgagaacc tggagatcaa cctgaggaag gagatcgcca 2940 aggcgttcaa gggcaacgag ggctacaagt cccttcaa gaggacacc atcgagacga 3000 tcctcccgga gttcctggac vakaaggacg agatcgccct ggtcaactcc ttcaacggct 3060 tcaccacggc gttcaccggc ttctcgaca accgcgagaa catgttcagc gaggaggcca 3120 agtccacgag catcgcgttc aggtgcatca acgagaacct cacccgctac atctccaaca 3180 tggacatctt cgagaggtc gacgagatct tcgacagca cgaggtgcag gagatgagg 3240 agaagagatcct gaacagcgac tacgacgtcg aggactctt cgaggggcgag ttcttcaact 3300 tcgtcctcac gcaggaggggc atcgacgtgt acacgccat catcggtggc ttcgtgaccg 3360 agtccggcga gagatcaag ggcctgaacg agtacatca cctctacaac cagaagacca 3420 agcagaagct gccgaagttc aagccccctgt acagcaggt gctctccgac agggagtccc 3480 tcagcttcta cggcgaggggc tacacgagcg acgaggaggt cctggaggtg ttccgcaaca 3540 ccctcaacaa gacagcgag atctctcca gcatcagaa gctcgagaag ctgttcaaga 3600 acttcgacga gtactccagc gccggcatct tcgtcaagaa cggcccggcg atctccacga 3660 tcagcaagga catcttcggc gagtggaacg tgatccgcga caagtggaac gccgagtacg 3720 acgacatcca cctcaagaag aaggcggtgg tcaccgagaa gtacgaggac gacaggcgca 3780 agtccttcaa gaagatcggc tccttcagcc tcgagcagct gcaggagtac gccgacgcgg 3840 acctgagcgt ggtcgagaag ctcaaggaga tcatcatcca gaaggtcgac gagatctaca 3900 aggtgtacgg ctccagcgag aagctcttcg acgcggactt cgtcctcgag aagtccctga 3960 agaagaacga cgccgtggtc gcgatcatga aggacctcct ggactccgtg aagagcttcg 4020 agaattacat caaggccttc ttcggcgagg gcaaggagac gaacagggac gagtccttct 4080 acggcgactt cgtcctggcc tacgacatcc tcctgaaggt ggaccacatc tacgacgcga 4140 tccgcaacta cgtgacccag aagccgtaca gcaaggacaa gttcaagctc tacttccaga 4200 acccccagtt catgggcggc tgggacaagg acaaggagac ggactacagg gcgaccatcc 4260 tgcgctacgg cagcaagtac tacctcgcca tcatggacaa gaagtacgcg aagtgcctgc 4320 agaagatcga caaggacgac gtcaacggca actacgagaa gatcaactac aagctcctgc 4380 cgggccccaa caagatgctc ccgaaggtgt tcttctccaa gaagtggatg gcctactaca 4440 accccagcga ggacatccag aagatctaca agaacggcac gttcaagaag ggcgacatgt 4500 tcaacctgaa cgactgccac aagctcatcg acttcttcaa ggactccatc agccgctacc 4560 cgaagtggtc caacgcctac gacttcaact tcagcgagac cgagaagtac aaggacatcg 4620 cgggcttcta ccgcgaggtc gaggagcagg gctacaaggt gtccttcgag tccgccagca 4680 agaaggaggt cgacaagctg gtggaggagg gcaagctcta catgttccag atctacaaca 4740 aggacttc cgacaagagc cacggcacgc ccaacctgca caccatgtac ttcaagctcc 4800 tgttcgacga gaacaaccac ggccagatca ggctgtccgg cggcgccgag ctcttcatga 4860 ggagggcgag cctgaagaag gaggagctgg tggtccaccc cgctaacagc caatcgcga 4920 acaagaaccc ggacaacccc aagaagacca cgaccctgtc ctacgacgtg tacaaggaca 4980 agaggttcag cgaggaccag tacgagctcc acatcccgat cgcgatcaac aagtgcccca 5040 agaacatctt caagatcaac accgaggtcc gcgtgctcct gaagcacgac gacaacccct 5100 acgtgatcgg catcgctagg ggcgagagga acctcctgta catcgtggtc gtggacggca 5160 agggcaacat cgtggagcag tactccctca acgagatcat caacaacttc aacggcatca 5220 ggatcaagac ggactaccac agcctcctgg acaagaagga gaaggagagg ttcgaggccc 5280 gccagaactg gacctccatc gagaacatca aggagctgaa ggcgggctac atcagccagg 5340 tcgtgcacaa gatctgcgag ctcgtcgaga agtacgacgc cgtgatcgcc ctcgcggacc 5400 tgaactccgg cttcaagaac agccgcgtca aggtggagaa gcaggtctac cagaagttcg 5460 agaagatgct catcgacaag ctgaactaca tggtggacaa gaagtccaac ccctgcgcta 5520 cgggcggcgc gctgaagggc taccagatca ccaacaagtt cgagagcttc aagtccatga 5580 gcactcagaa cggcttcatc ttctacatcc cggcgtggct cacgtccaag atcgacccca 5640 gcaccggctt cgtcaacctc ctgaagacga agtacacctc catcgccgac agcaagaagt 5700 tcatctccag cttcgaccgc atcatgtatg tgccggagga ggacctgttc gagtcgccc 5760 tcgactacaa gaacttctcc cgcacggacg cggactacat caagaagtgg aagctgtaca 5820 gctacggcaa ccgcatccgc atcttcagga accccaagaa gaacaacgtc ttcgactggg 5880 aggaggtgtg cctgacctcc gcgtacaagg agctcttcaa caagtacggc atcaactacc 5940 agcagggcga catcagggct ctcctgtgcg agcagagcga caaggccttc tactccagct 6000 tcatggcgct gatgtccctc atgctgcaga tgaggaactc gatcaccggc aggacggacg 6060 tggccttcct catctccccg gtgaagaaca gcgacggcat cttctacgac tccaggaact 6120 acgaggccca ggagaacgcg atcctcccaa agaacgcgga cgccaacggc gcctacaaca 6180 tcgccaggaa ggtcctctgg gctatcggcc agttcaagaa ggcggaggac gagaagctgg 6240 acaaggtgaa gatcgccatc agcaacaagg agtggctcga gtacgcccag acctcggtca 6300 agcacggcag cccgaagaag aagcgcaagg tgtccggcgg cagcacgaac ctgtccgaca 6360 tcatcgagaa ggagaccggc aagcagctcg tgatccagga gagcatcctc atgctgccgg 6420 aggaggtcga ggaggtcatc ggcaacaagc ccgagtccga catcctcgtc cacacggcct 6480 acgacgagtc caccgacgag aacgtgatgc tcctgacctc ggacgctccc gagtacaagc 6540 catgggccct ggtcatccag gacagcaacg gcgagaacaa gatcaagatg ctctccggcg 6600 gcagcccgaa gaagaagcgc aaagtgtgag atcgttcaaa catttggcaa taaagtttct 6660 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 6720 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 6780 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 6840 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tc 6882 <210> 10 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 10 gggggcgggg gctcaggcgg gggcgggagc ggcggcgggg gctctggggg cggcggcagc 60 ggcgggggcg gcagcggggg cggcgggtcg 90 <210> 11 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 11 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Gly Gly Ser Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Ser Gly <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic[[ID=3,9]] <400> 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 14 <211> 4842 0<212> DNA <213> Artificial Sequence <220> <223> Codon-optimized Fusion <400> 14 atgccgaaga agaagcgcaa ggtcatgtcc agcgagaccg gccccgtggc ggtggacccc 60 accctgcgca ggcgcatcga gccgcacgag ttcgaggtgt tcttcgaccc cagggagctc 120 cgcaaggaga cctgcctcct gtacgagatc aactggggcg gcaggcactc catctggagg 180 cacacgagcc agaacaccaa caagcacgtc gaggtgaact tcatcgagaa gttcaccacg 240 gagaggtact tctgcccgaa cacgcgctgc tccatcacgt ggttcctctc gtggagccca 300 tgcggcgagt gctccagggc gatcacggag ttcctcagcc gctacccgca cgtgaccctg 360 ttcatctaca tcgctaggct ctaccaccac gcggacccca ggaacaggca gggcctcagg 420 gacctgatct ccagcggcgt cacgatccag atcatgaccg agcaggagtc cggctactgc 480 tggaggaact tcgtgaacta ctccccgagc aacgaggccc actggccccg ctacccgcac 540 ctctgggtcc gcctctacgt gctcgagctg tactgcatca tcctcggcct gccgccctgc 600 ctcaacatcc tgaggcgcaa gcagccccag ctgacgttct tcaccatcgc cctgcagagc 660 tgccactacc agaggctccc gccccacatc ctgtgggcga ccgggctcaa gtcgggcagc 720 gagacccccg gcacctccga gtcggctacc ccagagtcca tgagcaagct ggagaagttc 780 acgaactgct actccctcag caagaccctg aggttcaagg cgatcccggt cggcaagacc 840 caggagaaca tcgacaaca gcggctgctg gtggaggacg agaagaggggc tgaggactac 900 aagggcgtga agaagctcct ggaccgctac tacctgtcct tcatcaacga cgtgctccac 960 agcatcaagc tcagaacct gacaacac atcagcctct tcaggagaa gacgcgcc 1020 gagagaga aaggagct cgagaacctg gagatchacc tgaggaagg gatcgccag 1080 gcgttcagg gcaacgagggg ctacaagtcc ctcttcaaga aggacat cgagacgatc 1140 ctcccggagt tcctggacga caggacgag atcgccctgg tcactcctt caacggctc 1200 accacggcgt tcaccggctt cttcgacaac cgcgagacaca tgttcagcga ggaggccaag 1260 tccacgagca tcgcgttcag gtgcatcaac gagaacctca cccgctacat ctccacatg 1320 gatacttcg agaggtcga cgcgatctc gatagcacg aggtgcagga gathaggag 1380 aagatcctga acagcgacta cgacgtcgag gactctctcg agggcgagtt cttcaacttc 1440 gtcctcacgc aggaggcat cgacgtgtac aacgccatca tcggtggctt cgtgaccgag 1500 tccggcgaga agatcaaggg cctgaacgag tacacaacc tctachaacca gagaccaag 1560 cagaagctgc cgaagttcaa gcccctgtac aagcaggtgc tctccgacag ggagtccctc 1620 agcttctacg gcgagggcta cacgagcgac gaggaggtcc tggaggtgtt ccgcaacacc 1680 ctcaacaaga acagcgagat cttctccagc atcaagaagc tcgagaagct gttcaagaac 1740 ttcgacgagt actccagcgc cggcatcttc gtcaagaacg gcccggcgat ctccacgatc 1800 1860 gacatccacc tcaagaagaa ggcggtggtc accgagaagt acgaggacga caggcgcaag 1920 tccttcaaga agatcggctc cttcagcctc gagcagctgc aggagtacg cgacgcggac 1980 ctgagcgtgg tcgagaagct caaggagatc atcatccaga aggtcgacga gatctacaag 2040 gtgtacggct ccagcgagaa gctcttcgac gcggacttcg tcctcgagaa gtccctgaag 2100 aagaacgacg ccgtggtcgc gatcatgaag gacctcctgg actccgtgaa gagcttcgag 2160 aattacatca aggccttctt cggcgagggc aaggacga acagggacga gtccttctac 2220 ggcgacttcg tcctggccta cgacatcctc ctgaaggtgg accacatcta cgacgcgatc 2280 cgcaactacg tgacccagaa gccgtacagc aagcaagt tcaagctcta cttccagaac 2340 ccccagttca tgggcggctg ggacaaggac aaggagacgg actacagggc gaccatcctg 2400 cgctacggca gcaagtacta cctcgccatc atggaaga agtacggaa gtgcctgcag 2460 aagatcgaca aggacgacgt caacggcaac tacgagaagaa tcaactacaa gctcctgccg 2520 ggccccaaca agatgctccc gaaggtgttc ttctccaaga agtggatggc ctactacaac 2580 cccagcgagg acatccagaa gatctacaag aacggcacgt tcaagaaggg cgacatgttc 2640 aacctgaacg actgccacaa gctcatcgac ttcttcaagg actccatcag ccgctacccg 2700 aagtggtcca acgcctacga cttcaacttc agcgagaccg agagtacaa ggacatcgcg 2760 ggcttctacc gcgaggttcga ggagcagggc tacaaggtgt ccttcgagtc cgccagcaag 2820 aaggaggtcg acaagctggt ggaggagggc aagctctaca tgttccagat ctacaacaag 2880 gacttctccg aaagagcca cggcacgccc aacctgcaca ccatgtactt caagctcctg 2940 ttcgacgaga acaccacgg ccagatcagg ctgtccggcg gcgccgagct cttcatgagg 3000 agggcgagcc tgaagaagga ggagctggtg gtccaccccg ctaacagccc aatcgcgaac 3060 aagaacccgg acaccacca gagaccacg accctgtcct acgacgtgta caggacaag 3120 aggttcagcg aggaccagta cgagctccac atcccgatcg cgatcaacaa gtgccccaag 3180 aacatcttca agatcacac cgaggtccgc gtgctcctga agcacgacga caaccctac 3240 gtgatcggca tcgctagggg cgagaggaac ctcctgtaca tcgtggtcgt ggacggcaag 3300 ggcacatcg tggagta ctcctcaac gagatcatca acaacttca cggcatcagg 3360 atcaagacgg actaccacag cctcctggac agaaggaga aggaggtt cgaggccccgc 3420 cagaactgga cctccatcga gaacatcag gagctgaagg cgggctacat cagccaggtc 3480 gtgcacaaga tctgcgagct cgtcgagaag tacgacgccg tgatcgccct cgcggacctg 3540 aactccggct tcagacag ccgcgtcaag gtggagaagc agtctacca gaagttcgag 3600 aagatgctca tcgacaagct gaactacatg gtggacaaga agtccaaccc ctgcgctacg 3660 ggcggcgcgc tgaagggcta ccagatcacc aacaagttcg agagcttcaa gtccatgagc 3720 actcagaacg gcttcatctt ctacatcccg gcgtggctca cgtccaagat cgaccccagc 3780 accggcttcg tcaacctcct gaaggaag tacacctcca tcgccgacag caagaagttc 3840 atctccagct tcgaccgcat catgtatgtg ccggaggagg acctgttcga gttcgccctc 3900 3960 tacggcaacc gcatccgcat cttcaggaac cccaagaaga acaacgtctt cgactgggag 4020 gaggtgtgcc tgacctccgc gtacaaggag ctcttcaaca agtacggcat caactaccag 4080 cagggcgaca tcagggctct cctgtgcgag cagagcgaca aggccttcta ctccagcttc 4140 atggcgctga tgtccctcat gctgcagatg aggaactcga tcaccggcag gacggacgtg 4200 gccttcctca tctccccggt gaaacagc gacggcatct tctacgactc caggaactac 4260 gaggcccagg agaacgcgat cctcccaaag aacgcggacg ccaacggcgc ctacaacatc 4320 gccaggaagg tcctctgggc tatcggccag ttcaagaagg cggagcacga gaagctggac 4380 aaggtgaga tcgccatcag caaaggag tggctcgagt acgcccagac ctcggtcaag 4440 cacggcagcc cgaagaagaa gcgcaaggtg ggagggtcga caggaggcgg ttctggcgga 4500 ggttcaggtg gaggctcgag tggtacgaac ctgtccgaca tcatcgagaa ggagaccggc 4560 aagcagctcg tgatccagga gagcatcctc atgctgccgg aggaggtcga ggaggtcatc 4620 ggcaacaagc ccgagtccga catcctcgtc cacacggcct acgacgagtc caccgacgag 4680 aacgtgatgc tcctgacctc ggacgctccc gagtacaagc catgggccct ggtcatccag 4740 gacagcaacg gcgagaacaa gatcaagatg ctcggtggag gcggttcagg cggaggtggc 4800 tctggcggtg gcggatcgcc gaagaagaag cgcaaagtgt ga 4842 <210> 15 <211> 1613 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 15 Met Pro Lys Lys Lys Arg Lys Val Met Ser Ser Glu Thr Gly Pro Val 1 5 10 15 Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu 20 25 30 Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr 35 40 45 Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln 50 55 60 Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr 65 70 75 80 Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu 85 90 95 Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu 100 105 110 Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr 115 120 125 His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser 130 135 140 Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys 145 150 155 160 Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro 165 170 175 Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys 180 185 190 Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln 195 200 205 Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln 210 215 220 Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser 225 230 235 240 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Ser Lys 245 250 255 Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 260 265 270 Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg 275 280 285 Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys 290 295 300 Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His 305 310 315 320 Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys 325 330 335 Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile 340 345 350 Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr 355 360 365 Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe 370 375 380 Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe 385 390 395 400 Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser 405 410 415 Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn 420 425 430 Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala 435 440 445 Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn 450 455 460 Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe 465 470 475 480 Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly 485 490 495 Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile 500 505 510 Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro 515 520 525 Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly 530 535 540 Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr 545 550 555 560 Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys 565 570 575 Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys 580 585 590 Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp 595 600 605 Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu 610 615 620 Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys 625 630 635 640 Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr 645 650 655 Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile 660 665 670 Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu 675,680,685 Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala 690,695,700 Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu 705 710 715 720 Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp 725 730 735 Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys 740,745,750 Val Asp His With Tyr Asp Only With Arg Asn Tyr Val Thr Gln Lys Pro 755,760,765 Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met 770,775,780 Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu 785,790,795,800 Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala 805 810 815 Lys Cys Leu Gln Lys Ile Asp Lys Asp Val Asn Gly Asn Tyr Glu 820 825 830 Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys 835 840 845 Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp 850 855 860 Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe 865 870 875 880 Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile 885,890,895 Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu 900 905 910 Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu 915,920,925 Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Glu Val Asp 930,935,940 Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys 945 950 955 960 Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr 965,970,975 Phe Lys Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser 980,985,990 Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu 995 1000 1005 Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro 1010 1015 1020 Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys 1025 1030 1035 Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 1040 1045 1050 Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu 1055 1060 1065 Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly 1070 1075 1080 Ile Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp 1085 1090 1095 Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile 1100 1105 1110 Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu 1115 1120 1125 Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp 1130 1135 1140 Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser 1145 1150 1155 Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala 1160 1165 1170 Val Ile Ala Leu Ala Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg 1175 1180 1185 Val Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu 1190 1195 1200 Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys 1205 1210 1215 Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe 1220 1225 1230 Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr 1235 1240 1245 Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe 1250 1255 1260 Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys 1265 1270 1275 Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu 1280 1285 1290 Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr 1295 1300 1305 Asp Ala Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr Gly Asn 1310 1315 1320 Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp 1325 1330 1335 Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn 1340 1345 1350 Lys Tyr Gly With Asn Tyr Gln Gly Asp With Arg Ala Leu Leu 1355 1360 1365 Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu 1370 1375 1380 Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr 1385 1390 1395 Asp Val Ala Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile 1400 1405 1410 Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu 1415 1420 1425 Pro Lys Asn Only Asp Only Asn Gly Only Tyr Asn Ile Only Arg Lys 1430 1435 1440 Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys 1445 1450 1455 Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu 1460 1465 1470 Tyr Ala Gln Thr Ser Val Lys His Gly Ser Pro Lys Lys Lys Arg 1475 1480 1485 Lys Val Gly Gly Ser Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly 1490 1495 1500 Gly Gly Ser Ser Gly Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu 1505 1510 1515 Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro 1520 1525 1530 Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile 1535 1540 1545 Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met 1550 1555 1560 Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val 1565 1570 1575 Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Gly Gly 1580 1585 1590 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Lys 1595 1600 1605 Lys Lys Arg Lys Val 1610 <210> 16 <211> 5145 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 16 atgccgaaga agaagcgcaa ggtcatgtcc agcgagaccg gccccgtggc ggtggacccc 60 accctgcgca ggcgcatcga gccgcacgag ttcgaggtgt tcttcgaccc cagggagctc 120 cgcaaggaga cctgcctcct gtacgagatc aactggggcg gcaggcactc catctggagg 180 cacacgagcc agaacaccaa caagcacgtc gaggtgaact tcatcgagaa gttcaccacg 240 gagaggtact tctgcccgaa cacgcgctgc tccatcacgt ggttcctctc gtggagccca 300 tgcggcgagt gctccagggc gatcacggag ttcctcagcc gctacccgca cgtgaccctg 360 ttcatctaca tcgctaggct ctaccaccac gcggacccca ggaacaggca gggcctcagg 420 gacctgatct ccagcggcgt cacgatccag atcatgaccg agcaggagtc cggctactgc 480 tggaggaact tcgtgaacta ctccccgagc aacgaggccc actggccccg ctacccgcac 540 ctctgggtcc gcctctacgt gctcgagctg tactgcatca tcctcggcct gccgccctgc 600 ctcaacatcc tgaggcgcaa gcagccccag ctgacgttct tcaccatcgc cctgcagagc 660 tgccactacc agaggctccc gccccacatc ctgtgggcga ccgggctcaa ggggggcggg 720 ggctcaggcg ggggcgggag cggcggcggg ggctctgggg gcggcggcag cggcggggc 780 ggcagcgggg gcggcgggtc gatgagcaag ctggagaagt tcacgaactg ctactccctc 840 agcaagaccc tgaggttcaa ggcgatcccg gtcggcaaga cccaggagaa catcgacaac 900 aagcggctgc tggtggagga cgagagaagg gctgagact acaagggcgt gaagaagctc 960 ctggaccgct actacctgtc cttcatcaac gacgtgctcc acagcatcaa gctcaagaac 1020 ctgaacaact acatcagcct cttcaggaag aagacgcgca ccgagaagga gaacaaggag 1080 ctcgagaacc tggagatcaa cctgaggaag gagatcgcca aggcgttcaa gggcaacgag 1140 ggctacaagt ccctcttcaa gaaggacatc atcgagacga tcctcccgga gttcctggac 1200 gacaaggacg agatcgccct ggtcaactcc ttcaacggct tcaccacggc gttcaccggc 1260 ttcttcgaca accgcgagaa catgttcagc gaggaggcca agtccacgag catcgcgttc 1320 aggtgcatca acgagaacct cacccgctac atctccaaca tggacatctt cgagaaggtc 1380 gacgcgatct tcgacaagca cgaggtgcag gagatcaagg agaagatcct gaacagcgac 1440 tacgacgtcg aggacttctt cgagggcgag ttcttcaact tcgtcctcac gcaggagggc 1500 atcgacgtgt acaacgccat catcggtggc ttcgtgaccg agtccggcga gaagatcaag 1560 ggcctgaacg agtacatcaa cctctacaac cagaagacca agcagaagct gccgaagttc 1620 aagcccctgt acaagcaggt gctctccgac agggagtccc tcagcttcta cggcgagggc 1680 tacacgagcg acgaggaggt cctggaggtg ttccgcaaca ccctcaacaa gaacagcgag 1740 atcttctcca gcatcaagaa gctcgagaag ctgttcaaga acttcgacga gtactccagc 1800 gccggcatct tcgtcaagaa cggcccggcg atctccacga tcagcaagga catcttcggc 1860 gagtggaacg tgatccgcga caagtggaac gccgagtacg acgacatcca cctcaagaag 1920 aaggcggtgg tcaccgagaa gtacgaggac gacaggcgca agtccttcaa gaagatcggc 1980 tccttcagcc tcgagcagct gcaggagtac gccgacgcgg acctgagcgt ggtcgagaag 2040 ctcaaggaga tcatcatcca gaaggtcgac gagatctaca aggtgtacgg ctccagcgag 2100 aagctcttcg acgcggactt cgtcctcgag aagtccctga agaagaacga cgccgtggtc 2160 gcgatcatga aggacctcct ggactccgtg aagagcttcg agaattacat caaggccttc 2220 ttcggcgagg gcaaggagac gaacagggac gagtccttct acggcgactt cgtcctggcc 2280 tacgacatcc tcctgaaggt ggaccacatc tacgacgcga tccgcaacta cgtgacccag 2340 aagccgtaca gcaaggacaa gttcaagctc tacttccaga acccccagtt catgggcggc 2400 tgggacaagg acaaggagac ggactacagg gcgaccatcc tgcgctacgg cagcaagtac 2460 tacctcgcca tcatggacaa gaagtacgcg aagtgcctgc agaagatcga caaggacgac 2520 gtcaacggca actacgagaa gatcaactac aagctcctgc cgggccccaa caagatgctc 2580 ccgaaggtgt tcttctccaa gaagtggatg gcctactaca accccagcga ggacatccag 2640 aagatctaca agaacggcac gttcaagaag ggcgacatgt tcaacctgaa cgactgccac 2700 aagctcatcg acttcttcaa ggactccatc agccgctacc cgaagtggtc caacgcctac 2760 gacttcaact tcagcgagac cgagaagtac aaggacatcg cgggcttcta ccgcgaggtc 2820 gaggagcagg gctacaaggt gtccttcgag tccgccagca agaaggaggt cgacaagctg 2880 gtggaggagg gcaagctcta catgttccag atctacaaca aggacttctc cgacaagagc 2940 cacggcacgc ccaacctgca caccatgtac ttcaagctcc tgttcgacga gaacaaccac 3000 ggccagatca ggctgtccgg cggcgccgag ctcttcatga ggagggcgag cctgaagaag 3060 gaggagctgg tggtccaccc cgctaacagc ccaatcgcga acaagaaccc ggacaacccc 3120 aagaagacca cgaccctgtc ctacgacgtg tacaaggaca agaggttcag cgaggaccag 3180 tacgagctcc acatcccgat cgcgatcaac aagtgcccca agaacatctt caagatcaac 3240 accgaggtcc gcgtgctcct gaagcacgac gacaacccct acgtgatcgg catcgctagg 3300 ggcgagagga acctcctgta catcgtggtc gtggacggca agggcaacat cgtggagcag 3360 tactccctca aacgagatcat cacaacttc aacggcatca ggatcagac ggactaccac 3420 agcctcctgg acagaagga gaaggagagg ttcgaggccc gccagaactg gacctccatc 3480 gagaacatca aggagctgaa ggcggctac atcagccagg tcgtgcaca gatctgcgag 3540 ctcgtcgaga agtacgacgc cgtgatcgcc ctcgcggacc tgaactccgg cttcagaac 3600 agccgcgtca aggtggagaa gcaggtctac cagaagttcg agagatgct catcgacaag 3660 ctgaactaca tggtggacaa gaagtccaac ccctgcgcta cggggcggcgc gctgaagggc 3720 taccagatca ccacaagtt cgagagcttc aagtccatga gcactcagaa cggcttcatc 3780 ttctacatcc cggcgtggct cacgtccaag atcgacccca gcaccggct cgtcaacctc 3840 ctgaagacga agtacaccctc catcgccgac agcagaagt tcatctccag cttcgaccgc 3900 atcatgtag tgccggagga ggacctgttc gagttcgccc tcgactaca gaacttctcc 3960 cgcacggacg cggactacat caagaagtgg aagctgtaca gctacggcaa ccgcatccgc 4020 atcttcagga acccagaa gaacaacgtc ttcgactggg aggaggtg cctgacctcc 4080 gcgtacaagg agctcttcaa caagtaccgg atcaactacc agcagggcga catcagggct 4140 ctcctgtgcg agcagagcga caaggcctt tactccagct tcatggcgct gatgtccctc 4200 atgctccaga tgaggaactc gatcaccggc aggacggacg tggccttcct catctccccg 4260 gtgaagaaca gcgacggcat cttctacgac tccaggaact acgaggccca ggagaacgcg 4320 atcctcccaa agaacgcgga cgccaacggc gcctacaaca tcgccaggaa ggtcctctgg 4380 gctatcggcc agttcaagaa ggcggagac gagaagctgg acaaggtgaa gatcgccatc 4440 4500 aagcgcaagg tgggagggtc gacaggaggc ggttctggcg gaggttcagg tggaggctcg 4560 agtggtacga acctgtccga catcatcgag aaggagaccg gcaagcagct cgtgatccag 4620 gagagcatcc tcatgctgcc ggaggaggtc gaggaggtca tcggcaacaa gcccgagtcc 4680 gacatcctcg tccacacggc ctacgacgag tccaccgacg agaacgtgat gctcctgacc 4740 tcggacgctc ccgagtacaa gccatgggcc ctggtcatcc aggacagcaa cggcgagaac 4800 aagatcaaga tgctcggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcg 4860 acgaacctgt ccgacatcat cgagaaggag accggcaagc agctcgtgat ccaggagagc 4920 atcctcatgc tgccggagga ggtcgaggag gtcatcggca acaagcccga gtccgacatc 4980 ctcgtccaca cggcctacga cgagtccacc gacgagaacg tgatgctcct gacctcggac 5040 gctcccgagt acaagccatg ggccctggtc atccaggaca gcaacggcga gaacaagatc 5100 aagatgctct ccggcggcag cccgaagaag aagcgcaaag tgtga 5145 <210> 17 <211> 1714 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Met Pro Lys Lys Lys Arg Lys Val Met Ser Ser Glu Thr Gly Pro Val 1 5 10 15 Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu 20 25 30 Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr 35 40 45 Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln 50 55 60 Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr 65 70 75 80 Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu 85 90 95 Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu 100 105 110 Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr 115 120 125 His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser 130 135 140 Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys 145 150 155 160 Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro 165 170 175 Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys 180 185 190 Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln 195 200 205 Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln 210 215 220 Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Lys Leu Glu 260 265 270 Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala 275 280 285 Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu 290 295 300 Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu 305 310 315 320 Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile 325 330 335 Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr 340 345 350 Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu 355 360 365 Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser 370 375 380 Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp 385 390 395 400 Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr 405 410 415 Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu 420 425 430 Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr 435 440 445 Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe 450 455 460 Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp 465 470 475 480 Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu 485 490 495 Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val 500 505 510 Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu 515 520 525 Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr 530 535 540 Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly 545 550 555 560 Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn 565 570 575 Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe 580 585 590 Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly 595 600 605 Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val 610 615 620 Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys 625 630 635 640 Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe 645 650 655 Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp 660 665 670 Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys 675 680 685 Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp 690,695,700 Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Ala Val Val 705 710 715 720 Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr 725 730 735 Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser 740,745,750 Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp 755,760,765 His Tyr Asp Has Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser 770,775,780 Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly 785,790,795,800 Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr With Leu Arg Tyr 805 810 815 Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys 820 825 830 Leu Gln Lys Ile Asp Lys Asp Val Asn Gly Asn Tyr Glu Lys Ile 835 840 845 Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe 850 855 860 Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln 865 870 875 880 Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu 885 890 895 Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg 900 905 910 Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu 915 920 925 Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly 930 935 940 Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu 945 950 955 960 Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe 965 970 975 Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys 980 985 990 Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly 995 1000 1005 Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu 1010 1015 1020 Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp 1025 1030 1035 Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp 1040 1045 1050 Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala 1055 1060 1065 Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val 1070 1075 1080 Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile 1085 1090 1095 Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly 1100 1105 1110 Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn 1115 1120 1125 Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu 1130 1135 1140 Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr 1145 1150 1155 Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln 1160 1165 1170 Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val 1175 1180 1185 Ile Ala Leu Ala Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val 1190 1195 1200 Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile 1205 1210 1215 Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala 1220 1225 1230 Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu 1235 1240 1245 Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile 1250 1255 1260 Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val 1265 1270 1275 Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys 1280 1285 1290 Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp 1295 1300 1305 Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp 1310 1315 1320 Only Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg 1325 1330 1335 Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp 1340 1345 1350 Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys 1355 1360 1365 Tyr Gly With Asn Tyr Gln Gln Gly Asp With Arg Ala Leu Leu Cys 1370 1375 1380 Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met 1385 1390 1395 Serve Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp 1400 1405 1410 Val Ala Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe 1415 1420 1425 Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro 1430 1435 1440 Lys Asn Only Asp Only Asn Gly Only Tyr Asn Only Only Arg Lys Val 1445 1450 1455 Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu 1460 1465 1470 Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr 1475 1480 1485 Ala Gln Thr Ser Val Lys His Gly Ser Pro Lys Lys Lys Arg Lys 1490 1495 1500 Val Gly Gly Ser Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 1505 1510 1515 Gly Ser Ser Gly Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr 1520 1525 1530 Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu 1535 1540 1545 Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu 1550 1555 1560 Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu 1565 1570 1575 Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile 1580 1585 1590 Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Gly Gly Gly 1595 1600 1605 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asn Leu 1610 1615 1620 Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln 1625 1630 1635 Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly 1640 1645 1650 Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu 1655 1660 1665 Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu 1670 1675 1680 Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn 1685 1690 1695 Lys Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys 1700 1705 1710 Val <210> 18 <211> 4767 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 18 atgccgaaga agaagcgcaa ggtcatgtcc agcgagaccg gccccgtggc ggtggacccc 60 accctgcgca ggcgcatcga gccgcacgag ttcgaggtgt tcttcgaccc cagggagctc 120 cgcaaggaga cctgcctcct gtacgagatc aactggggcg gcaggcactc catctggagg 180 cacacgagcc agaacaccaa caagcacgtc gaggtgaact tcatcgagaa gttcaccacg 240 gagaggtact tctgcccgaa cacgcgctgc tccatcacgt ggttcctctc gtggagccca 300 tgcggcgagt gctccagggc gatcacggag ttcctcagcc gctacccgca cgtgaccctg 360 ttcatctaca tcgctaggct ctaccaccac gcggacccca ggaacaggca gggcctcagg 420 gacctgatct ccagcggcgt cacgatccag atcatgaccg agcaggagtc cggctactgc 480 tggaggaact tcgtgaacta ctccccgagc aacgaggccc actggccccg ctacccgcac 540 ctctgggtcc gcctctacgt gctcgagctg tactgcatca tcctcggcct gccgccctgc 600 ctcaacatcc tgaggcgcaa gcagccccag ctgacgttct tcaccatcgc cctgcagagc 660 tgccactacc agaggctccc gccccacatc ctgtgggcga ccgggctcaa gtcgggcagc 720 gagacccccg gcacctccga gtcggctacc ccagagtcca tgagcaagct ggagaagttc 780 acgaactgct actccctcag cagaccctg aggttcagg cgatcccggt cggcaagacc 840 caggagaaca tcgacaaca gcggctgctg gtggaggacg agaagaggggc tgaggactac 900 aagggcgtga agaagctcct ggaccgctac tacctgtcct tcatcaacga cgtgctccac 960 agcatcaagc tcagaacct gacaacac atcagcctct tcaggagaa gacgcgcc 1020 gagagaga aaggagct cgagaacctg gagatchacc tgaggaagg gatcgccag 1080 gcgttcagg gcaacgagggg ctacaagtcc ctcttcaaga aggacat cgagacgatc 1140 ctcccggagt tcctggacga caggacgag atcgccctgg tcactcctt caacggctc 1200 accacggcgt tcaccggctt cttcgacaac cgcgagacaca tgttcagcga ggaggccaag 1260 tccacgagca tcgcgttcag gtgcatcaac gagaacctca cccgctacat ctccacatg 1320 gatacttcg agaggtcga cgcgatctc gatagcacg aggtgcagga gathaggag 1380 aagatcctga acagcgacta cgacgtcgag gactctctcg agggcgagtt cttcaacttc 1440 gtcctcacgc aggaggcat cgacgtgtac aacgccatca tcggtggctt cgtgaccgag 1500 tccgggggaga agatcaaggg cctgaacgag tacatcaacc tctacaacca gaaccaag 1560 cagaagctgc cgaagttcaa gcccctgtac aagcaggtgc tctccgacag ggagtccctc 1620 agcttctacg gcgagggcta cacgagcgac gaggaggtcc tggaggtgtt ccgcaacacc 1680 ctcaacaaga acagcgagat cttctccagc atcaagaagc tcgagaagct gttcaagaac 1740 ttcgacgagt actccagcgc cggcatcttc gtcaagaacg gcccggcgat ctccacgatc 1800 1860 gacatccacc tcaagaagaa ggcggtggtc accgagaagt acgaggacga caggcgcaag 1920 tccttcaaga agatcggctc cttcagcctc gagcagctgc aggagtacg cgacgcggac 1980 ctgagcgtgg tcgagaagct caaggagatc atcatccaga aggtcgacga gatctacaag 2040 gtgtacggct ccagcgagaa gctcttcgac gcggacttcg tcctcgagaa gtccctgaag 2100 aagaacgacg ccgtggtcgc gatcatgaag gacctcctgg actccgtgaa gagcttcgag 2160 aattacatca aggccttctt cggcgagggc aaggacga acagggacga gtccttctac 2220 ggcgacttcg tcctggccta cgacatcctc ctgaaggtgg accacatcta cgacgcgatc 2280 cgcaactacg tgacccagaa gccgtacagc aagcaagt tcaagctcta cttccagaac 2340 ccccagttca tgggcggctg ggacaaggac aaggagacgg actacagggc gaccatcctg 2400 cgctacggca gcaagtacta cctcgccatc atggaaga agtacggaa gtgcctgcag 2460 aagatcgaca aggacgacgt caacggcaac tacgagaagaa tcaactacaa gctcctgccg 2520 ggccccaaca agatgctccc gaaggtgttc ttctccaaga agtggatggc ctactacaac 2580 cccagcgagg acatccagaa gatctacaag aacggcacgt tcaagaaggg cgacatgttc 2640 aacctgaacg actgccacaa gctcatcgac ttcttcaagg actccatcag ccgctacccg 2700 aagtggtcca acgcctacga cttcaacttc agcgagaccg agagtacaa ggacatcgcg 2760 ggcttctacc gcgaggttcga ggagcagggc tacaaggtgt ccttcgagtc cgccagcaag 2820 aaggaggtcg acaagctggt ggaggagggc aagctctaca tgttccagat ctacaacaag 2880 gacttctccg aaagagcca cggcacgccc aacctgcaca ccatgtactt caagctcctg 2940 ttcgacgaga acaaccacgg ccagatcagg ctgtccggcg gcgccgagct cttcatgagg 3000 agggcgagcc tgaagaagga ggagctggtg gtccaccccg ctaacagccc aatcgcgaac 3060 aagaacccgg acaccacca gagaccacg accctgtcct acgacgtgta caggacaag 3120 aggttcagcg aggaccagta cgagctccac atcccgatcg cgatcaacaa gtgccccaag 3180 aacatcttca agatcacac cgaggtccgc gtgctcctga agcacgacga caaccctac 3240 gtgatcggca tcgctagggg cgagaggaac ctcctgtaca tcgtggtcgt ggacggcaag 3300 ggcacatcg tggagta ctcctcaac gagatcatca acaacttca cggcatcagg 3360 atcaagacgg actaccacag cctcctggac agaaggaga aggaggtt cgaggccccgc 3420 cagaactgga cctccatcga gaacatcag gagctgaagg cgggctacat cagccaggtc 3480 gtgcacaaga tctgcgagct cgtcgagaag tacgacgccg tgatcgccct cgcggacctg 3540 aactccggct tcagacag ccgcgtcaag gtggagaagc agtctacca gaagttcgag 3600 aagatgctca tcgacaagct gaactacatg gtggacaaga agtccaaccc ctgcgctacg 3660 ggcggcgcgc tgaagggcta ccagatcacc aacaagttcg agagcttcaa gtccatgagc 3720 actcagaacg gcttcatctt ctacatcccg gcgtggctca cgtccaagat cgaccccagc 3780 accggcttcg tcaacctcct gaaggaag tacacctcca tcgccgacag caagaagttc 3840 atctccagct tcgaccgcat catgtatgtg ccggaggagg acctgttcga gttcgccctc 3900 3960 tacggcaacc gcatccgcat cttcaggaac cccaagaaga acaacgtctt cgactgggag 4020 gaggtgtgcc tgacctccgc gtacaaggag ctcttcaaca agtacggcat caactaccag 4080 cagggcgaca tcagggctct cctgtgcgag cagagcgaca aggccttcta ctccagcttc 4140 atggcgctga tgtccctcat gctgcagatg aggaactcga tcaccggcag gacggacgtg 4200 gccttcctca tctccccggt gaaacagc gacggcatct tctacgactc caggaactac 4260 gaggcccagg agaacgcgat cctcccaaag aacgcggacg ccaacggcgc ctacaacatc 4320 gccaggaagg tcctctgggc tatcggccag ttcaagaagg cggagcacga gaagctggac 4380 aaggtgaaga tcgccatcag caacaaggag tggctcgagt acgcccagac ctcggtcaag 4440 cacggcagcc cgaagaagaa gcgcaaggtg tccggcggca gcacgaacct gtccgacatc 4500 atcgagaagg agaccggcaa gcagctcgtg atccaggaga gcatcctcat gctgccggag 4560 gaggtcgagg aggtcatcgg caacaagccc gagtccgaca tcctcgtcca cacggcctac 4620 gacgagtcca ccgacgagaa cgtgatgctc ctgacctcgg acgctcccga gtacaagcca 4680 tgggccctgg tcatccagga cagcaacggc gagaacaaga tcaagatgct ctccggcggc 4740 agcccgaaga agaagcgcaa agtgtga 4767 <210> 19 <211> 1588 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 19 Met Pro Lys Lys Lys Arg Lys Val Met Ser Ser Glu Thr Gly Pro Val 1 5 10 15 Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu 20 25 30 Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr 35 40 45 Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln 50 55 60 Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr 65 70 75 80 Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu 85 90 95 Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu 100 105 110 Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr 115 120 125 His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser 130 135 140 Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys 145 150 155 160 Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro 165 170 175 Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys 180 185 190 Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln 195 200 205 Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln 210 215 220 Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser 225 230 235 240 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Ser Lys 245 250 255 Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 260 265 270 Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg 275 280 285 Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys 290 295 300 Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His 305 310 315 320 Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys 325 330 335 Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile 340 345 350 Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr 355 360 365 Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe 370 375 380 Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe 385 390 395 400 Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser 405 410 415 Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn 420 425 430 Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala 435 440 445 Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn 450 455 460 Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe 465 470 475 480 Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly 485 490 495 Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile 500 505 510 Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro 515 520 525 Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly 530 535 540 Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr 545 550 555 560 Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys 565 570 575 Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys 580 585 590 Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp 595 600 605 Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu 610 615 620 Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys 625 630 635 640 Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr 645 650 655 Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile 660 665 670 Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu 675,680,685 Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala 690,695,700 Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu 705 710 715 720 Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp 725 730 735 Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys 740,745,750 Val Asp His With Tyr Asp Only With Arg Asn Tyr Val Thr Gln Lys Pro 755,760,765 Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met 770,775,780 Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu 785,790,795,800 Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala 805 810 815 Lys Cys Leu Gln Lys Ile Asp Lys Asp Val Asn Gly Asn Tyr Glu 820 825 830 Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys 835 840 845 Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp 850 855 860 Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe 865 870 875 880 Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile 885,890,895 Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu 900 905 910 Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu 915,920,925 Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Glu Val Asp 930,935,940 Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys 945 950 955 960 Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr 965,970,975 Phe Lys Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser 980,985,990 Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu 995 1000 1005 Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro 1010 1015 1020 Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys 1025 1030 1035 Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 1040 1045 1050 Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu 1055 1060 1065 Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly 1070 1075 1080 Ile Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp 1085 1090 1095 Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile 1100 1105 1110 Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu 1115 1120 1125 Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp 1130 1135 1140 Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser 1145 1150 1155 Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala 1160 1165 1170 Val Ile Ala Leu Ala Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg 1175 1180 1185 Val Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu 1190 1195 1200 Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys 1205 1210 1215 Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe 1220 1225 1230 Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr 1235 1240 1245 Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe 1250 1255 1260 Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys 1265 1270 1275 Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu 1280 1285 1290 Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr 1295 1300 1305 Asp Ala Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr Gly Asn 1310 1315 1320 Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp 1325 1330 1335 Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn 1340 1345 1350 Lys Tyr Gly With Asn Tyr Gln Gly Asp With Arg Ala Leu Leu 1355 1360 1365 Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu 1370 1375 1380 Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr 1385 1390 1395 Asp Val Ala Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile 1400 1405 1410 Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu 1415 1420 1425 Pro Lys Asn Only Asp Only Asn Gly Only Tyr Asn Ile Only Arg Lys 1430 1435 1440 Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys 1445 1450 1455 Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu 1460 1465 1470 Tyr Ala Gln Thr Ser Val Lys His Gly Ser Pro Lys Lys Lys Arg 1475 1480 1485 Lys Val Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys 1490 1495 1500 Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu 1505 1510 1515 Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp 1520 1525 1530 Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val 1535 1540 1545 Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu 1550 1555 1560 Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser 1565 1570 1575 Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1580 1585 <210> 20 <211> 5229 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 20 atgccgaaga agaagcgcaa ggtgtccagc gagaccggcc ccgtggcggt cgaccccacc 60 ctgcgcaggc gcatcgagcc gcacgagttc gaggtcttct tcgaccccag ggagctccgc 120 aaggagacct gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac 180 accagccaga acacgaacaa gcacgtggag gtcaacttca tcgagaagtt caccacggag 240 aggtacttct gcccgaacac ccgctgctcc atcacctggt tcctctcgtg gagcccatgc 300 ggcgagtgct ccagggcgat cacggagttc ctcagccgct acccgcacgt gaccctcttc 360 atctacatcg ctaggctgta ccaccacgcg gaccccagga acaggcaggg gctcagggac 420 ctgatctcca gcggcgtgac catccagatc atgacggagc aggagtccgg ctactgctgg 480 cgcaacttcg tcaactactc cccgagcaac gaggcccact ggccccgcta cccgcacctg 540 tgggtgcgcc tctacgtcct cgagctgtac tgcatcatcc tcggcctgcc gccctgcctc 600 aacatcctga ggcgcaagca gccccagctc accttcttca cgatcgccct gcagagctgc 660 cactaccagc ggctgccgcc ccacatcctc tgggccaccg gcctgaagtc gggcagcgag 720 acgcccggca cgtccgagtc ggctacccca gagctcaagg acaagagta cagcatcggc 780 ctggcaatcg gcaccaacag cgtgggctgg gccgtgatca ccgacgagta caaggtgccg 840 agcaagaagt tcaaggtgct gggcaacacc gacaggcaca gcatcaaga gaacctgatc ggcgccctgc tgttcgacag cggcgagacc gccgaggcca ccaggctgaa gaggaccgcc 960 aggaggaggt acaccaggag gaagacagg atctgctacc tgcaggagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacaggctgg aggagagctt cctggtggag 1080 gaggacaaga agcacgagag gcacccgatc ttcggcaaca tcgtggacga ggtggcctac 1140. 1200. acccgaccat ctaccacctg aggaagagc tggtggacag caccgacag gccgacctga ggctgatcta cctggccctg gcccacatga tcaagttcag gggccacttc 1260 ctgatcgagg gcgacctgaa cccggacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggag aacccgatca acgccagcgg cgtggacgcc 1380 aaggccatcc tgagcgccag gctgagcaag agcaggaggc tggagaacct gatcgcccag 1440 ctgccgggcg agaagaagaa cggcctgttc ggcaacctga tcgccctgag cctgggcctg 1500 accccgaact tcaagagcaa cttcgacctg gccgaggacg ccaagctgca gctgagcaag 1560 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac 1620 ctgttcctgg ccgccaagaa cctgagcgac gccatcctgc tgagcgacat cctgagggtg 1680 aacaccgaga tcaccaaggc cccgctgagc gccagcatga tcaagaggta cgaccagcac 1740 caccaggacc tgaccctgct gaaggccctg gtgaggcagc agctgccgga gaagtacaag 1800 gagatcttct tcgaccagag caagaacggc tacgccggct acatcgacgg cggcgccagc 1860 caggaggagt tctacaagtt catcaagccg atcctggaga agatggacgg caccgaggag 1920 ctgctggtga agctgaacag ggaggacctg ctgaggaagc agaggacctt cgacaacggc 1980 agcatcccgc accagatcca cctgggcgag ctgcacgcca tcctgaggag gcaggaggac 2040 ttctacccgt tcctgaagga caacagggag aagatcgaga agatcctgac cttccgcatc 2100 ccgtactacg tgggcccgct ggccaggggc aacagcaggt tcgcctggat gaccaggaag 2160 agcgaggaga ccatcacccc gtggaacttc gaggaggtgg tggacaaggg cgccagcgcc 2220 cagagcttca tcgagaggat gaccaacttc gacaagaacc tgccgaacga gaaggtgctg 2280 ccgaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac caaggtgaag 2340 tacgtgaccg agggcatgag gaagccggcc ttcctgagcg gcgagcagaa gaaggccatc 2400 gtggacctgc tgttcaagac caacaggaag gtgaccgtga agcagctgaa ggaggactac 2460 ttcaagaaga tcgagtgctt cgacagcgtg gagatcagcg gcgtggagga caggttcaac 2520 gccagcctgg gcacctacca cgacctgctg aagatcatca aggacaagga cttcctggac 2580 aacgaggaga acgaggacat cctggaggac atcgtgctga ccctgaccct gttcgaggac 2640 agggagatga tcgaggagag gctgaagacc tacgcccacc tgttcgacga caaggtgatg 2700 aagcagctga agaggaggag gtacaccggc tggggcaggc tgagcaggaa gctgatcaac 2760 ggcatcaggg acagcagag cggcagacc atcctggact tcctgaagg cgacggctc 2820 gccaacagga acttcatgca gctgatccac gacgacagcc tgaccttca ggaggacatc 2880 cagaaggccc aggtgagcgg ccaggggcgac agcctgcacg agcacatcgc caacctggcc 2940 ggcagcccgg ccatcaagaa gggcatcctg cagaccgtga aggtggtgga cgagctggtg 3000 aaggtgatgg gcaggcacaa gccggagac atcgtgatcg agatggccag ggagaaccag 3060 accacccaga agggccagaa gacagcagg gagagatga agaggatcga ggaggcatc 3120 aaggagctgg gcagccagat cctgaggg cacccggtgg agacaccca gctgcagaac 3180 gagaagctgt acctgtacta cctgcagaac ggcagggaca tgtacgtgga ccaggagctg 3240 gatacaaca ggctgagcga ctacgacgtg gaccacatcg tgccgcagag cttcctgaag 3300 gacgacagca tcgahacaa ggtgctgacc aggacgac agacagggg gagagcgac 3360 aacgtgccga gcgaggaggt ggtgagaag atgaaaact actggaggca gctgctgaac 3420 gccaagctga tcaccagag gaagttcgac aacctgacca agggccgag gggcggcctg 3480 agcgagctgg acaaggccgg cttcattaaa aggcagctgg tggagaccag gcagatcacc 3540 aagcacgtgg cccagatcct ggacagcagg atgaacacca agtacgacga gaacgacaag 3600 ctgatcaggg aggtgaaggt gatcaccctg aagagcaagc tggtgagcga cttcaggaag 3660 gacttccagt tctacaaggt gagggagatc aataattacc accacgccca cgacgcctac 3720 ctgaacgccg tggtgggcac cgccctgatt aaaaagtacc cgaagctgga gagcgagttc 3780 gtgtacggcg actacaaggt gtacgacgtg aggaagatga tcgccaagag cgagcaggag 3840 atcggcaagg ccaccgccaa gtacttcttc tacagcaaca tcatgaactt cttcaagacc 3900 gagatcaccc tggccaacgg cgagatcagg aagaggccgc tgatcgagac caacggcgag 3960 accggcgaga tcgtgtggga caagggcagg gacttcgcca ccgtgaggaa ggtgctgtcc 4020 atgccgcagg tgaacatcgt gaagaagacc gaggtgcaga ccggcggctt cagcaaggag 4080 agcatcctgc cgaaggaa cagcgacaag ctgatcgcca ggagaaagga ctgggatccc 4140 aagaagtcg gcggcttcga cagcccgacc gtggcctaca gcgtgctggt ggtggccaag 4200 gtgggaagg gcaagagcaa gaagctgaag agcgtgaagg agctggtggg catcaccatc 4260 atggagagga gcagcttcga gaagaaccca gtggacttcc tggaggccaa gggctacaag 4320 gaggtga aggacctgat cattaaactg ccgaagtaca gcctgttcga gctggagaac 4380 ggcaggaga ggatgctggc cagcgccggc gagctgcaga agggcaacga gctggccctg 4440 ccgagcaagt acgtgaactt cctgtacctg gccagccact acgagaagct gaagggcagc 4500 ccgggagaca acgagcagaa gcagctgttc gtggagcagc acaagcacta cctggacgag 4560 atcatcgagc agatcagcga gttcagcaag agggtgatcc tggccgacgc caacctggac 4620 aaggtgctga gcgcctacaa caagcacagg gacaagccga tcagggagca ggccgagaac 4680 atcatccacc tgttcaccct gaccaacctg ggcgccccgg ccgccttcaa gtacttcgac 4740 accaccatcg acaggaagag gtacaccagc accaaggagg tgctggacgc caccctgatc 4800 caccagagca tcaccggcct gtacgagacc aggatcgacc tgagccagct gggcggcgac 4860 agcagcccgc cgaagagaa gagggaggtg agctgggaagg acgccagcgg ctggagcagg 4920 atgaccaggg actccggcgg cagcaccaac ctctccgaca tcatcgagaa ggagacgggc 4980 aagcagctcg tgatccagga gagcatcctc atgctgccgg aggaggtgga ggaggtcatc 5040 ggcaacaagc ccgagtccga catcctcgtg cacacggcct acgacgagtc caccgacgag 5100 aacgtcatgc tcctgacctc ggacgctccc gagtacaagc catgggccct cgtgatccag 5160 gacagcaacg gcgagaacaa gatcaagatg ctctccggcg gcagcccgaa gaagaagcgc 5220 aaagtctga 5229 <210> 21 <211> 1742 <%212> PRT<%213> Artificial sequence <220> <223> Synthetic <400> 21 Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala 1 5 10 15 Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val 20 25 30 Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu 35 40 45 Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn 50 55 60 It should be noted that there may be some inaccuracies in the translation of specific terms in the patent text, and it is recommended to further consult relevant professional materials for accurate understanding.Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu 65 70 75 80 Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser 85 90 95 Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser 100 105 110 Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His 115 120 125 His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser 130 135 140 Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp 145 150 155 160 Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg 165 170 175 Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile 180 185 190 Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro 195 200 205 Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg 210 215 220 Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu 225 230 235 240 Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Asp Lys Lys 245 250 255 Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val 260 265 270 Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly 275 280 285 Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu 290 295 300 Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala 305 310 315 320 Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu 325 330 335 Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg 340 345 350 Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His 355 360 365 Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr 370 375 380 Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys 385 390 395 400 Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe 405 410 415 Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp 420 425 430 Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe 435 440 445 Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu 450 455 460 Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln 465 470 475 480 Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu 485 490 495 Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu 500 505 510 Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp 515 520 525 Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala 530 535 540 Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val 545 550 555 560 Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg 565 570 575 Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg 580 585 590 Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys 595 600 605 Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe 610 615 620 Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu 625 630 635 640 Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr 645 650 655 Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His 660 665 670 Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn 675 680 685 Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val 690 695 700 Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys 705 710 715 720 Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys 725 730 735 Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys 740 745 750 Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu 755 760 765 Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu 770 775 780 Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile 785 790 795 800 Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu 805 810 815 Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile 820 825 830 Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp 835 840 845 Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn 850 855 860 Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp 865 870 875 880 Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp 885 890 895 Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly 900 905 910 Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly 915 920 925 Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn 930 935 940 Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile 945 950 955 960 Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile 965 970 975 Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr 980 985 990 Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro 995 1000 1005 Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 1010 1015 1020 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu 1025 1030 1035 Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val 1040 1045 1050 Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 1055 1060 1065 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn 1070 1075 1080 Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe 1085 1090 1095 Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp 1100 1105 1110 Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val 1115 1120 1125 Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu 1130 1135 1140 Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly 1145 1150 1155 Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 1160 1165 1170 Glu Thr Arg Gln Ile Thr Lys His Valve Gln Ile Leu Asp 1175 1180 1185 Arg Met Thr Lys Tyr Asp Glu Asn Asp Lys With Arg 1190 1195 1200 Glu Val Lys Val Ile Thr Leu Lys For Lys Leu Val For Asp Phe 1205 1210 1215 Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr 1220 1225 1230 His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala 1235 1240 1245 Leu Glu Ser Glu Phe Val Tyr Gly 1250 1255 1260 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu 1265 1270 1275 Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn 1280 1285 1290 I Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1295 1300 1305 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu 1310 1315 1320 Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val 1325 1330 1335 Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1340 1345 1350 Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser 1355 1360 1365 Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr 1370 1375 1380 Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val 1385 1390 1395 Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys 1400 1405 1410 Glu Leu Val Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys 1415 1420 1425 Asn Pro Val Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys 1430 1435 1440 Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu 1445 1450 1455 Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln 1460 1465 1470 Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu 1475 1480 1485 Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1490 1495 1500 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu 1505 1510 1515 Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile 1520 1525 1530 Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1535 1540 1545 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His 1550 1555 1560 Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr 1565 1570 1575 Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1580 1585 1590 Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr 1595 1600 1605 Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Ser Pro 1610 1615 1620 Pro Lys Lys Lys Arg Lys Val Ser Trp Lys Asp Ala Ser Gly Trp 1625 1630 1635 Ser Arg Met Thr Arg Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp 1640 1645 1650 Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser 1655 1660 1665 Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys 1670 1675 1680 Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr 1685 1690 1695 Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys 1700 1705 1710 Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile 1715 1720 1725​​​​​​​​​​​​ <400> 22 Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln 20 25 30 Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys 35 40 45 Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln 50 55 60 Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile 65 70 75 80 Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile 85 90 95 Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly 100 105 110 Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile 115 120 125 Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys 130 135 140 Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg 145 150 155 160 Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg 165 170 175 Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg 180 185 190 Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe 195 200 205 Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn 210 215 220 Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val 225 230 235 240 Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp 245 250 255 Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu 260 265 270 Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn 275 280 285 Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro 290 295 300 Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu 305 310 315 320 Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr 325 330 335 Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu 340 345 350 Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His 355 360 365 Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr 370 375 380 Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys 385 390 395 400 Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu 405 410 415 Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser 420 425 430 Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala 435 440 445 Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys 450 455 460 Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu 465 470 475 480 Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe 485 490 495 Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser 500 505 510 Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val 515 520 525 Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp 530 535 540 Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn 545 550 555 560 Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys 565 570 575 Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys 580 585 590 Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys 595 600 605 Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr 610 615 620 Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys 625 630 635 640 Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln 645 650 655 Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala 660 665 670 Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr 675 680 685 Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr 690 695 700 Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His 705 710 715 720 Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu 725 730 735 Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys 740 745 750 Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu 755 760 765 Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln 770 775 780 Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His 785 790 795 800 Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr 805 810 815 Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His 820 825 830 Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn 835 840 845 Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe 850 855 860 Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln 865 870 875 880 Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu 885 890 895 Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Ala Arg Gly Glu Arg 900 905 910 Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu 915 920 925 Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu 930 935 940 Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val 945 950 955 960 Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile 965 970 975 His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu 980 985 990 Ala Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu 995 1000 1005 Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu 1010 1015 1020 Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly 1025 1030 1035 Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala 1040 1045 1050 Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro 1055 1060 1065 Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe 1070 1075 1080 Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu 1085 1090 1095 Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe 1100 1105 1110 Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly 1115 1120 1125 Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn 1130 1135 1140 Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys 1145 1150 1155 Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr 1160 1165 1170 Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu 1175 1180 1185 Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu 1190 1195 1200 Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu 1205 1210 1215 Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly 1220 1225 1230 Glu Ala Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys 1235 1240 1245 Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp 1250 1255 1260 Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu 1265 1270 1275 Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile 1280 1285 1290 Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Gly 1295 1300 1305 Ser Pro Lys Lys Lys Arg Lys Val 1310 1315 <210> 23 <211> 4809 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 23 atgccgaaga agaagcgcaa ggtcatgtcc agcgagaccg gccccgtggc ggtggacccc 60 accctgcgca ggcgcatcga gccgcacgag ttcgaggtgt tcttcgaccc cagggagctc 120 cgcaaggaga cctgcctcct gtacgagatc aactggggcg gcaggcactc catctggagg 180 cacacgagcc agaacaccaa caagcacgtc gaggtgaact tcatcgagaa gttcaccacg 240 gagaggtact tctgcccgaa cacgcgctgc tccatcacgt ggttcctctc gtggagccca 300 tgcggcgagt gctccagggc gatcacggag ttcctcagcc gctacccgca cgtgaccctg 360 ttcatctaca tcgctaggct ctaccaccac gcggacccca ggaacaggca gggcctcagg 420 gacctgatct ccagcggcgt cacgatccag atcatgaccg agcaggagtc cggctactgc 480 tggaggaact tcgtgaacta ctccccgagc aacgaggccc actggccccg ctacccgcac 540 ctctgggtcc gcctctacgt gctcgagctg tactgcatca tcctcggcct gccgccctgc 600 ctcaacatcc tgaggcgcaa gcagccccag ctgacgttct tcaccatcgc cctgcagagc 660 tgccactacc agaggctccc gccccacatc ctgtgggcga ccgggctcaa ggggggcggg 720 ggctcaggcg ggggcgggag cggcggcggg ggctctgggg gcggcggcag cggcggggc 780 ggcagcgggg gcggcgggtc gatgagcaag ctggagaagt tcacgaactg ctactccctc 840 agcaagaccc tgaggttcaa ggcgatcccg gtcggcaaga cccaggagaa catcgacaac 900 aagcggctgc tggtggagga cgagagaagg gctgagact acaagggcgt gaagaagctc 960 ctggaccgct actacctgtc cttcatcaac gacgtgctcc acaccatca gctcagaac 1020 ctgaacaact acatcagcct cttcaggaag aagacgcgca ccgaagga gaacaggag 1080 ctcgagaacc tggagatcaa cctgaggaag gagatcgcca aggcttca gggcaacgag 1140 ggctacaagt ccccttcaa gaaggacatc atcgagacga tcctcccgga gttcctggac 1200 ngaaggacg agatcgccct gtcaactcc ttcaacggct tcaccacggc gttcaccggc 1260 ttctcgaca accgcgagaa catgttcagc gaggaggcca agtccacgag catcgcgttc 1320 aggtgcatca acgagaacct cacccgctac atctccaca tggacatctt cgagaaggtc 1380 gacgcgatct tcgacaagca cgaggtgcag gagatcagg agagatcct gaacagcgac 1440 tacgacgtcg aggactctt cgaggggcgag ttctcact tcgtcctcac gcaggagggc 1500 atcgacgtgt acacgccat catcggtggc ttcgtgaccg agtccggcga gagatcaag 1560 ggcctgaacg agtacatca cctctacac cagagacca agcagaagct gccgaagttc 1620 aagcccctgt acaagcaggt gctctccgac agggagtccc tcagctctta cggcgaggc 1680 tacacgagcg acgaggaggt cctggaggtg ttccgcaaca ccctcaacaa gaacagcgag 1740 atcttctcca gcatcaagaa gctcgagaag ctgttcaaga acttcgacga gtactccagc 1800 gccggcatct tcgtcaagaa cggcccggcg atctccacga tcagcaagga catcttcggc 1860 gagtggaacg tgatccgcga caagtggaac gccgagtacg acgacatcca cctcaagaag 1920 aaggcggtgg tcaccgagaa gtacgaggac gacaggcgca agtccttcaa gaagatcggc 1980 tccttcagcc tcgagcagct gcaggagtac gccgacgcgg acctgagcgt ggtcgagaag 2040 ctcaaggaga tcatcatcca gaaggtcgac gagatctaca aggtgtacgg ctccagcgag 2100 aagctcttcg acgcggactt cgtcctcgag aagtccctga agaagaacga cgccgtggtc 2160 gcgatcatga aggacctcct ggactccgtg aagagcttcg agaattacat caaggccttc 2220 ttcggcgagg gcaaggagac gaacagggac gagtccttct acggcgactt cgtcctggcc 2280 tacgacatcc tcctgaaggt ggaccacatc tacgacgcga tccgcaacta cgtgacccag 2340 aagccgtaca gcaaggacaa gttcaagctc tacttccaga acccccagtt catgggcggc 2400 tgggacaagg acaggagac ggactacagg gcgaccatcc tgcgctacgg cagcaagtac 2460 tacctcgcca tcatggacaa gaagtacgcg aagtgcctgc agaagatcga caaggacgacgac 2520 gtcaacggca actacgagaa gatcaactac aagctcctgc cgggccccaa caagatgctc 2580 ccgaaggtgt tcttctccaa gaagtggatg gcctactaca accccagcga ggacatccag 2640 aagatctaca agaacggcac gttcaagaag ggcgacatgt tcaacctgaa cgactgccac 2700 aagctcatcg acttcttcaa ggactccatc agccgctacc cgaagtggtc caacgcctac 2760 gacttcaact tcagcgagac cgagaagtac areacatcg cgggcttcta ccgcgaggtc 2820 gaggagcagg gctacaaggt gtccttcgag tccgccagca agaaggaggt cgacaagctg 2880 gtggaggagg gcaagctcta catgttccag atctacaaca aggacttctc cgacaagagc 2940 cacggcacgc ccaacctgca caccatgtac ttcaagctcc tgttcgacga gaacaccac 3000 ggccagatca ggctgtccgg cggcgccgag ctcttcatga ggagggcgag cctgaagaag 3060 gaggagctgg tggtccaccc cgctaacagc ccaatcgcga acaagaaccc ggacaacccc 3120 aagaagacca cgaccctgtc ctacgacgtg tacaaggaca agaggttcag cgaggaccag 3180 tacgagctcc acatcccgat cgcgatcaac aagtgcccca agaacatctt caagatcaac 3240 accgaggtcc gcgtgctcct gaagcacgac gacaacccct acgtgatcgg catcgctagg 3300 ggcgagagga acctcctgta catcgtggtc gtggacggca agggcaacat cgtggagcag 3360 tactccctca acgagatcat caacaacttc aacggcatca ggatcaagac ggactaccac 3420 agcctcctgg acaagaagga gaaggagagg ttcgaggccc gccagaactg gacctccatc 3480 gagaacatca aggagctgaa ggcgggctac atcagccagg tcgtgcacaa gatctgcgag 3540 ctcgtcgaga agtacgacgc cgtgatcgcc ctcgcggacc tgaactccgg cttcaagaac 3600 agccgctca aggtggagaa gcaggtctac cagaagttcg agaagatgct catcgacaag 3660 ctgaactaca tggtggacaa gaagtccaac ccctgcgcta cgggcggcgc gctgaagggc 3720 taccagatca ccaacaagtt cgagagcttc aagtccatga gcactcagaa cggcttcatc 3780 ttctacatcc cggcgtggct cacgtccaag atcgacccca gcaccggctt cgtcaacctc 3840 ctgaagacga agtacacctc catcgccgac agcaagaagt tcatctccag cttcgaccgc 3900 atcatgtatg tgccggagga ggacctgttc gagttcgccc tcgactacaa gaacttctcc 3960 cgcacggacg cggactacat caagaagtgg aagctgtaca gctacggcaa ccgcatccgc 4020 atcttcagga accccaagaa gaacaacgtc ttcgactggg aggaggtgtg cctgacctcc 4080 gcgtacaagg agctcttcaa caagtacggc atcaactacc agcagggcga catcagggct 4140 ctcctgtgcg agcagagcga caaggccttc tactccagct tcatggcgct gatgtccctc 4200 atgctgcaga tgaggaactc gatcaccggc aggacggacg tggccttcct catctccccg 4260 gtgaagaaca gcgacggcat cttctacgac tccaggaact acgaggccca ggagaacgcg 4320 atcctcccaa agaacgcgga cgccaacggc gcctacaaca tcgccaggaa ggtcctctgg 4380 gctatcggcc agttcaagaa ggcggaggac gagaagctgg acaaggtgaa gatcgccatc 4440 agcaacaagg agtggctcga gtacgcccag acctcggtca agcacggcag cccgaagaag 4500 aagcgcaagg tgtccggcgg cagcacgaac ctgtccgaca tcatcgagaa ggagaccggc 4560 aagcagctcg tgatccagga gagcatcctc atgctgccgg aggaggtcga ggaggtcatc 4620 ggcaacaagc ccgagtccga catcctcgtc cacacggcct acgacgagtc caccgacgag 4680 aacgtgatgc tcctgacctc ggacgctccc gagtacaagc catgggccct ggtcatccag 4740 gacagcaacg gcgagaacaa gatcaagatg ctctccggcg gcagcccgaa gaagaagcgc 4800 aaagtgtga 4809 <210> 24 <211> 1602 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 24 Met Pro Lys Lys Lys Arg Lys Val Met Ser Ser Glu Thr Gly Pro Val 1 5 10 15 Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu 20 25 30 Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr 35 40 45 Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln 50 55 60 Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr 65 70 75 80 Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu 85 90 95 Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu 100 105 110 Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr 115 120 125 His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser 130 135 140 Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys 145 150 155 160 Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro 165 170 175 Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys 180 185 190 Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln 195 200 205 Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln 210 215 220 Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Lys Leu Glu 260 265 270 Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala 275 280 285 Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu 290 295 300 Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu 305 310 315 320 Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile 325 330 335 Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr 340 345 350 Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu 355 360 365 Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser 370 375 380 Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp 385 390 395 400 Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr 405 410 415 Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu 420 425 430 Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr 435 440 445 Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe 450 455 460 Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp 465 470 475 480 Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu 485 490 495 Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val 500 505 510 Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu 515 520 525 Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr 530 535 540 Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly 545 550 555 560 Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn 565 570 575 Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe 580 585 590 Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly 595 600 605 Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val 610 615 620 Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys 625 630 635 640 Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe 645 650 655 Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp 660 665 670 Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys 675 680 685 Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp 690 695 700 Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Ala Val Val 705 710 715 720 Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr 725 730 735 Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser 740,745,750 Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp 755,760,765 His Tyr Asp Has Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser 770,775,780 Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly 785,790,795,800 Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr With Leu Arg Tyr 805 810 815 Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys 820 825 830 Leu Gln Lys Ile Asp Lys Asp Val Asn Gly Asn Tyr Glu Lys Ile 835 840 845 Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe 850 855 860 Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln 865 870 875 880 Lys Ile Tyr Lys Asn Gly Thr Phe Lys Gly Asp Met Phe Asn Leu 885,890,895 Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg 900 905 910 Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu 915,920,925 Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly 930,935,940 Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Glu Val Asp Lys Leu 945 950 955 960 Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe 965,970,975 Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys 980,985,990 Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly 995 1000 1005 Leu Glu Glu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu 1010 1015 1020 Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp 1025 1030 1035 Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp 1040 1045 1050 Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala 1055 1060 1065 Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val 1070 1075 1080 Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile 1085 1090 1095 Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly 1100 1105 1110 Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn 1115 1120 1125 Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu 1130 1135 1140 Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr 1145 1150 1155 Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln 1160 1165 1170 Val Val His Lys Ile Cys Glu Lion Val Glu Lys Tyr Asp Ala Val 1175 1180 1185 Ile Ala Leu Ala Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val 1190 1195 1200 Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile 1205 1210 1215 Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala 1220 1225 1230 Thr Gly Gly Ala Leu Lys Gly Tyr Gln With Thr Asn Lys Phe Glu 1235 1240 1245 Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile 1250 1255 1260 Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val 1265 1270 1275 Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys 1280 1285 1290 Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp 1295 1300 1305 Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp 1310 1315 1320 Only Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg 1325 1330 1335 Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp 1340 1345 1350 Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys 1355 1360 1365 Tyr Gly With Asn Tyr Gln Gln Gly Asp With Arg Ala Leu Leu Cys 1370 1375 1380 Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met 1385 1390 1395 Serve Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp 1400 1405 1410 Val Ala Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe 1415 1420 1425 Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro 1430 1435 1440 Lys Asn Only Asp Only Asn Gly Only Tyr Asn Only Only Arg Lys Val 1445 1450 1455 Leu Trp Ala Ile Gly Gln Phe Lys Ala Glu Asp Glu Lys Leu 1460 1465 1470 Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr 1475 1480 1485 Ala Gln Thr Ser Val Lys His Gly Ser Pro Lys Lys Lys Arg Lys 1490 1495 1500 Val Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu 1505 1510 1515 Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro 1520 1525 1530 Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile 1535 1540 1545 Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met 1550 1555 1560 Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val 1565 1570 1575 Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly 1580 1585 1590 Gly Ser Pro Lys Lys Lys Arg Lys Val 1595 1600 <210> 25 <211> 1802 <212> DNA <213> Sugarcane (Saccharum officinarum) <400> 25 gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 acgagaccgc gacgcaaccg cctctcgccg ctgggcccca caccgctcgg tgccgtagcc 300 tcacgggact ctttctccct cctccccgt tataaattgg cttcatcccc tccttgcctc 360 atccatccaa atcccagtcc ccaatcccat cccttcgtag gagaaattca tcgaagctaa 420 gcgaatcctc gcgatcctct caaggtactg cgagttttcg atccccctct cgacccctcg 480 tatgtttgtg tttgtcgtag cgtttgatta ggtatgcttt ccctgtttgt gttcgtcgta 540 gcgtttgatt aggtatgctt tccctgttcg tgttcatcgt agtgtttgat taggtcgtgt 600 gaggcgatgg cctgctcgcg tccttcgatc tgtagtcgat ttgcgggtcg tggtgtagat 660 ctgcgggctg tgatgaagtt atttggtgtg atctgctcgc ctgattctgc gggttggctc 720 gagtagatat gatggttgga ccggttggtt cgtttaccgc gctagggttg ggctgggatg 780 atgttgcatg cgccgttgcg cgtgatcccg cagcaggact tgcgtttgat tgccagatct 840 cgttacgatt atgtgatttg gtttggactt tttagatctg tagcttctgc ttatgtgcca 900 gatgcgccta ctgctcatat gcctgatgat aatcataaat ggctgtggaa ctaactagtt 960 gattgcggag tcatgtatca gctacaggtg tagggactag ctacaggtgt agggacttgc 1020 gtctaattgt ttggtccttt actcatgttg caattatgca atttagttta gattgtttgt 1080 tccactcatc taggctgtaa aagggacact gcttagattg ctgtttaatc tttttagtag 1140 attatattat attggtaact tattacccct attacatgcc atacgtgact tctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttg attgttgaat catgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac tgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaa gaatcaggtt 1380 cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat atatcactgg 1440 aattaattag ttgatcgttt aatcatatat caagtacata ccatgccaca atttttagtc 1500 acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgtt ctattctgat 1560 tagaccatat atcatgtatt tttttttggt aatggttctc ttattttaaa tgctatatag 1620 ttctggtact tgttagaaag atctgcttca tagtttagtt gcctatccct cgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaa ttcttttgtg ttcaacagtc 1740 agtttttgtt agattcattg taacttatgg tcgcttactc ttctggtcct caatgcttgc 1800[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ gtttggggaa agaccgagga gaagatctac gggcctgtcg ctggaacgga ctacagggac 60 aaccagctgc ggttcagcct gctatgccag 90 <210> 29 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 29 agatgggaga cgggtacgag acgg 24 <210> 30 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 30 gtatgggttg ttgttgaggc tcagg 25 <210> 31 <211> 25 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 31 gaccacccac tgttcctgga gaggg 25 <210> 32 <211> 3783 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 32 atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc ttcaaagctc 60 gagaattca ccaactgtta ttcgttgagc aaaacactgc ggtttaaagc gattccagtc 120 ggcaagactc aagagaatat agacaataag cggctgttgg tggagaatga aaagcgcgcg 180 gaagactaca aaggggtgaa gaagttgttg gaagatact acctctcttt tatcaatgat 240 gtcttgcact caatcaaatt gaagaatctg aacaactaca tctccctctt cagaaagaaa 300 360 degrees atcgcgaaag cctttaaagg caacgaagga tacaaaagtc tgttcaagaa ggatataatt 420 gagacaattt tgccagagtt cctcgatgac area ttgcgctggt caattcgttc 480 aacggattca caacagcatt cacaggcttc tttgataatc gggaaaatat gttctctgag 540 gaggcaaagt ccacttctat tgcgttcagg tgtatcaatg agaatctcac taggtacatt 600 660 attaagga agatcctcaa ttccgattat gacgttgagg acttcttcga gggtgagttt 720 tttaatttcg tgctcactca agagggtatc gacgtgtata atgcgatcat cggtgggttc 780 gtgactgagt ccggtgaaaa gattaaggga ttgaacgagt atcaacct ttacaaccaa 840 aagacgaaac agaagctgcc aaagttcaag cctctca aacaggttct ttcagaccgc 900 gagtcactct cgttctatgg ggaggctac acttcggatg aggagtcct ggaggtgttc 960 aggaatactc tcataagaa ttcggatt ttctctctta taaaaaaact ggaaaagttg 1020 tttaagaatt ttgacgaata ctctagcgcc ggcatatttg tgaaaaacgg cccggccata 1080 tcaacgataa gtaagatat cttcggcgaa tggaacgtga tcagagacaa atggaacgcg 1140 gagtatgacg attackcacct gagagaag gctgtcgtaa cggaagta cgaggatgat 1200 cgcaggaaaa gcttcaaaa gatcggaagt ttcagcctgg aacagttgca ggagtatgct 1260 gacgccgatc ttagcgtcgt cgagaagttg aaggaata tcatccaaaa ggtcgacgag 1320 atatataaag tctatggatc aagtgaaaaa ctgttcgacg ccgacttcgt tttggagaag 1380 tccctgaaga agaacgacgc tgttgttgcc attatgaagg atctgctcga cagcgtgaag 1440 agtttcgaga actatattaa ggctttttc ggggagggga aggagacta cagagatgag 1500 tccttctacg gagacttcgt cctcgcgtac gatatactcc tttaggtaga ccacatctac 1560 gacgcaatca gaaattacgt gacaaaag ccgtacagca aggacagtt cavacctac 1620 ttccagaacc cccagttcat gggcggctgg ngaaggaca aggaaacgga ttacaggggct 1680 acgatcctga ggtatggttc aaatactac ttggcgatta tggacaagaa gtacgccaag 1740 tgtctccaga agattgacaa agacgatgtc aatggcaatt atgagaat caactacaag 1800 ctgcttccgg gtccgaaca gatgctccca aaggttttct tcagcaaga atggatggcc 1860 tactatacc caagcgagga catchcagaag attattaga acggtacgtt cagaagggc 1920 gatagttca attackacga ctgtcacaag ctgatcgact tctcaagc ctcattagc 1980 cggtacccaa agtggtctaa cgcctatgac ttcaactttt cggaaccga gaagtacaag 2040 gatatagccg gattttatag agaggtggaa gagcagggct acaggtgtc attcgagtcc 2100 gccagcaaga aggaagtgga caagctcgtg gaagagggta agctctacat gttccagatt 2160 tataataag actttagcga taagagccac gggacaccta atctccacac aatgtatttc 2220 aagctgctct tcgacgagaa taaccacggc caaatcaggt tgtcaggagg ggctgaactc 2280 ttcatgcggc gcgctagcct taagaaggag gagcttgtag tccaccctgc gaatagtcca 2340 attgcgaata agaacccgga caatcctaaa aagactacaa cattgagcta cgacgtgtac 2400 your area ggttttccga ggatcagtac gagctccaca tcccgattgc gatcaacaag 2460 tgcccaaaga atatttcaa gataaacaca gaggtgcgtg tactcctgaa gcatgacgac 2520 aatccttacg tcattgggat tgatcggggc gagaggaacc tcctctatat tgtggtggtg 2580 gacgggaagg ggaacatagt cgaacagtac tcccttaacg aataattaa caatttcaac 2640 2700 gaggcgcggc aaaattggac aagtatcgag aacatcaagg aactcaaagc aggttatatc 2760 tctcaagttg tgcataagat atgcgagctg gttgagaagt atgacgcagt gatcgctctt 2820 gaggacctca actcgggctt taagaattct agagttaaag tggagaagca ggtctatcaa 2880 aagttcgaga agatgcttat agataagctc aactacatgg tcgataagaa atcgaaccca 2940 tgtgccaccg gcggcgcact caaaggttac caaataacaa acaaattcga gtccttcaaa 3000 tcgatgagta ctcagaatgg gttcatattt tatataccgg cgtggcttac gtctaagatc 3060 gacccgtcaa ctggttttgt caacctgttg aagacgaaat acacgtccat tgccgattca 3120 aaaaagttca tatctagttt tgatcgtatt atgtacgtcc cagaggaaga tcttttcgag 3180 tttgctctcg actacaaaaa cttttcgcgc accgatgcgg attacattaa aaaatggaaa 3240 ctctattcgt acggcaacag aatcaggatt tttcgcaacc ctaagaagaa taacgtcttt 3300 gattgggagg aagtttgctt gactagcgcg tacaagggac tctttaataa gtatggcatt 3360 aactaccaac agggtgatat cagagcactg ctttgcgaac aatctgacaa ggctttctac 3420 tcatccttca tggctttgat gagcctgatg ctccagatga gaaattcaat tacaggcaga 3480 accgacgtgg atttcttgat ctccccggtt aaaaattctg atggcatctt ttacgatagc 3540 aggaactatg aagcgcaaga gaatgcgatt ctgccaaaaa atgcagacgc caacggtgcc 3600 tataacatcg ccaggaaagt cctgtgggcg atcggccagt tcaaaaaggc cgaagacgaa 3660 aaattggaca aggtcaaaat cgctatcagc aacaaagagt ggctggagta tgctcagaca 3720 tccgtaaagc ataagcgtcc tgctgccacc aaaaaggccg gacaggctaa gaaaaagaag 3780 tga 3783 <210> 33 <211> 1260 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 33 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr 20 25 30 Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp 35 40 45 Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys 50 55 60 Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp 65 70 75 80 Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu 85 90 95 Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn 100 105 110 Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn 115 120 125 Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu 130 135 140 Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe 145 150 155 160 Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn 165 170 175 Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile 180 185 190 Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys 195 200 205 Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys 210 215 220 Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe 225 230 235 240 Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile 245 250 255 Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn 260 265 270 Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys 275 280 285 Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser 290 295 300 Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe 305 310 315 320 Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys 325 330 335 Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile 340 345 350 Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe 355 360 365 Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp 370 375 380 Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp 385 390 395 400 Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu 405 410 415 Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu 420 425 430 Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser 435 440 445 Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys 450 455 460 Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys 465 470 475 480 Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr 485,490,495 Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile 500 505 510 Leu Leu Lys Val Asp His With Tyr Asp With Arg Asn Tyr Val Thr 515,520,525 Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro 530 535 540 Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala 545 550 555 560 Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys 565,570,575 Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly 580 585 590 Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met 595 600 605 Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro 610 615 620 Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly 625 630 635 640 Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys 645 650 655 Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn 660 665 670 Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu 675 680 685 Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys 690 695 700 Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile 705 710 715 720 Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His 725 730 735 Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile 740 745 750 Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys 755 760 765 Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys 770 775 780 Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr 785 790 795 800 Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 805 810 815 Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val 820 825 830 Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp 835 840 845 Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly 850 855 860 Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn 865 870 875 880 Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu 885 890 895 Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile 900 905 910 Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys 915 920 925 Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn 930 935 940 Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln 945 950 955 960 Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys 965 970 975 Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile 980 985 990 Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe 995 1000 1005 Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser 1010 1015 1020 Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala 1025 1030 1035 Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val 1040 1045 1050 Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe 1055 1060 1065 Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser 1070 1075 1080 Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn 1085 1090 1095 Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu 1100 1105 1110 Leu Phe Asn Lys Tyr Gly And Asn Tyr Gln Gln Gly Asp With Arg 1115 1120 1125 Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe 1130 1135 1140 Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr 1145 1150 1155 Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser 1160 1165 1170 Asp Gly Has Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn 1175 1180 1185 Only Leu Pro Lys Asn Only Asp Only Asn Gly Only Tyr Asn Only 1190 1195 1200 Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu 1205 1210 1215 Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu 1220 1225 1230 Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro Ala 1235 1240 1245 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1250 1255 1260 <210> 34 <211> 3873 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 34 atgccgaaga agaagcgcaa ggtcgggggc gggggctcag gcgggggcgg gagcggcggc 60 gggggctctg ggggcggcgg cagcggcggg ggcggcagcg ggggcggcgg gtcgatgagc 120 aagctggaga agttcacgaa ctgctactcc ctcagcaaga ccctgaggtt caaggcgatc 180 ccggtcggca agacccagga gaacatcgac aacaagcggc tgctggtgga ggacgagaag 240 agggctgagg actacaaggg cgtgaagaag ctcctggacc gctactacct gtccttcatc 300 aacgacgtgc tccacaccat caagctcaag aacctgaaca actacatcag cctcttcagg 360 aagagacgc gcaccgagaa ggagacaag gagctcgaga acctggagat cacctgagg 420 aaggagatcg ccaaggcgtt caagggcac gaggctaca agtcccttt ccaaggac 480 atcatcgaga cgatcctccc ggagttcctg gacgacaagg acgagatcgc cctggtcaac 540 tccttcaacg gctcaccac ggcgttcacc ggctcttcg acaccgcga gaacatgttc 600 agcgaggagg ccaagtccac gagcatcgcg ttcaggtgca tcaacgagaa cctcaccccgc 660 tacatctcca acatggacat cttcgagaag gtcgacgcga tcttcgacaa gcacgaggtg 720 caggagatca aggagaat cctgaacagc gactacgacg tcgactt cttcgaggggc 780 gagttcttca acttcgtcct cacgcaggag ggcatcgacg tgtacaacgc catcatcggt 840 ggcttcgtga ccgagtccgg cgagaagagatc aagggcctga acgagtacat caacctac 900 aaccagaaga ccaagcagaa gctgccgaag ttcaagcccc tgtacaagca ggtgctctcc 960 ggagggagt ccctcagctt ctacggcgag ggctacacga gcgacgagga ggtcctggag 1020 gtgttccgca acaccctcaa caagaacagc gagatcttct ccagcatcaa gaagctcgag 1080 aagctgttca agaacttcga cgagtactcc agcgccggca tcttcgtcaa gaacggcccg 1140 gcgatctcca cgatcagcaa ggacatcttc ggcgagtgga acgtgatccg cgacaagtgg 1200 aacgccgagt acgacgacat ccacctcaag aagaaggcgg tggtcaccga gaagtacgag 1260 gacgacaggc gcaagtcctt caagaagatc ggctccttca gcctcgagca gctgcaggag 1320 tacgccgacg cggacctgag cgtggtcgag aagctcaagg agatcatcat ccagaaggtc 1380 gacgagatct acaaggtgta cggctccagc gagaagctct tcgacgcgga cttcgtcctc 1440 gagaagtccc tgaagaagaa cgacgccgtg gtcgcgatca tgaaggacct cctggactcc 1500 gtgaagagct tcgagaatta catcaaggcc ttcttcggcg agggcaagga gacgaacagg 1560 gacgagtcct tctacggcga cttcgtcctg gcctacgaca tcctcctgaa ggtggaccac 1620 atctacgacg cgatccgcaa ctacgtgacc cagaagccgt acagcaagga caagttcaag 1680 ctctacttcc agaaccccca gttcatgggc ggctgggaca aggacaagga gacggactac 1740 agggcgacca tcctgcgcta cggcagcaag tactacctcg ccatcatgga aagagtac 1800 gcgaagtgcc tgcagaagat cgacaaggac gacgtcaacg ccaactacga gaagtcaac 1860 tacaagctcc tgccgggccc caacaagatg ctcccgaagg tgttcttctc caagaagtgg 1920 atggcctact acaaccccag cgaggacatc cagaagatct acaagaacgg cacgttcaag 1980 aagggcgaca tgttcaacct gaacgactgc cacaagctca tcgacttctt caaggactcc 2040 atcagccgct acccgaagtg gtccaacgcc tacgacttca acttcagcga gaccgagaag 2100 tacaggaca tcgcgggctt ctaccgcgag gtcgaggagc agggctacaa ggtgtccttc 2160 gagtccgcca gcaagagga ggtcgacaag ctggtggagg agggcaagct ctacatgttc 2220 cagatctaca acaagactt ctccgacaag agccacggca cgcccaacct gcacaccatg 2280 tacttcaagc tcctgttcga cgagaacaac cagggccaga tcaggctgtc cggcggcgcc 2340 gagctcttca tgaggagggc gagcctgaag aaggaggac tggtggtcca ccccgctaac 2400 agcccaatcg cgaacaagaa cccggacaac cccaagaagaa ccacgaccct gtcctacgac 2460 gtgtacaagg acaagaggtt cagcgaggac cagtacgagc tccacatccc gatcgcgatc 2520 aacaagtgcc ccaagaacat cttcaagatc aacaccgagg tccgcgtgct cctgaagcac 2580 gacgacaacc cctacgtgat cggcatcgac aggggcgaga ggaacctcct gtacatcgtg 2640 gtcgtggacg gcaagggcaa catcgtggag cagtactccc tcaacgagat catcaacaac 2700 ttcaacggca tcaggatcaa gacggactac cacagcctcc tggacaagaa ggagaaggag 2760 aggttcgagg cccgccagaa ctggacctcc atcgagaaca tcaaggagct gaaggcgggc 2820 tacatcagcc aggtcgtgca caagatctgc gagctcgtcg agaagtacga cgccgtgatc 2880 gccctcgagg acctgaactc cggcttcaag aacagccgcg tcaaggtgga gaagcaggtc 2940 taccagaagt tcgagaagat gctcatcgac aagctgaact acatggtgga caagaagtcc 3000 aacccctgcg ctacgggcgg cgcgctgaag ggctaccaga tcaccaacaa gttcgagagc 3060 ttcaagtcca tgagcactca gaacggcttc atcttctaca tcccggcgtg gctcacgtcc 3120 aagatcgacc ccagcaccgg cttcgtcaac ctcctgaaga cgaagtacac ctccatcgcc 3180 gawgcaaga agttcatctc cagcttcgac cgcatcatgt atgtgccgga ggaggacctg 3240 ttcgagttcg ccctcgacta caaacttc tcccgcacgg acgcggacta catchagaag 3300 tggaagctgt acagctacgg siaccgcatc cgcatctca ggaaccccaa gagaacaac 3360 gtcttcgact gggaggaggt gtgcctgacc tccgcgtaca aggagctt cacaagtac 3420 ggcatcact accagcagggg cgacatcagg gctctcctgt gcgagcagag cgacaaggcc 3480 ttctactcca gcttcatggc gctgatgtcc ctcatgctgc agatgaggaa ctcgatcacc 3540 ggcaggacgg acgtggactt cctcatctcc ccggtgaaga acagcgacgg catcttac 3600 gactccagga actacgaggc ccaggagaac gcgatcctcc caagaacgc ggacgccac 3660 ggcgcctaca acatcgccag gaggtcctc tggctatcg gccagttca gaaggcggag 3720 gacgagaagc tggacaggt gagatcgcc atcagcaca aggagtggct cgagtacgcc 3780 cagacctcgg tcaagcacgg cagcccgaag aagaagcgca aggtgtccgg cggcagctcc 3840 ggcggcagcc cgagagaa gcgcaagtg tga 3873 <210> 35 <211> 1290 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 35 Met Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Ser Met Ser Lys Leu Glu Lys Phe Thr Asn Cys 35 40 45 Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys 50 55 60 Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys 65 70 75 80 Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr 85 90 95 Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu 100 105 110 Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu 115 120 125 Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala 130 135 140 Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp 145 150 155 160 Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile 165 170 175 Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe 180 185 190 Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser Thr Ser 195 200 205 Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn 210 215 220 Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu Val 225 230 235 240 Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp 245 250 255 Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile 260 265 270 Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu 275 280 285 Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr 290 295 300 Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser 305 310 315 320 Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu 325 330 335 Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile 340 345 350 Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu 355 360 365 Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr 370 375 380 Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp 385 390 395 400 Asn Ala Glu Tyr Asp Asp Ile His Leu Light Light Light Ala Val Val Thr 405 410 415 Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser 420 425 430 Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val 435 440 445 Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr 450 455 460 Lys Val Tyr Gly Ser Ser Glu Lys Phe Asp Ala Asp Phe Val Leu 465 470 475 480 Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp 485,490,495 Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe 500 505 510 Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe 515,520,525 Val Leu Ala Tyr Asp Ile Leu Lys Val Asp His Ile Tyr Asp Ala 530 535 540 With Arg Asn Tyr Val Thr Gln Pro Tyr Ser Lys Asp Lys Phe Lys 545 550 555 560 Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys 565,570,575 Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr 580,585,590 Leu Wing Ile Met Asp Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp 595,600,605 Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu 610 615 620 Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp 625 630 635 640 Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn 645 650 655 Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys 660 665 670 Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser 675 680 685 Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile 690 695 700 Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe 705 710 715 720 Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys 725 730 735 Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His 740 745 750 Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu 755 760 765 Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met 770 775 780 Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn 785 790 795 800 Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr 805 810 815 Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr 820 825 830 Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe 835 840 845 Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp Asn Pro 850 855 860 Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val 865 870 875 880 Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu 885 890 895 Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser 900 905 910 Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp 915,920,925 Thr Ser With Glu Asn With Lys Glu Leu Lys Ala Gly Tyr With Ser Gln 930,935,940 Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Isle 945 950 955 960 Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val 965,970,975 Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu 980,985,990 Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala 995 1000 1005 Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser 1010 1015 1020 Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu 1025 1030 1035 Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys 1040 1045 1050 Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser 1055 1060 1065 Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe 1070 1075 1080 Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile 1085 1090 1095 Lys Lys Trp Lys Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe 1100 1105 1110 Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys 1115 1120 1125 Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn 1130 1135 1140 Tyr Gln Gln Gly Asp With Arg Ala Leu Leu Cys Glu Gln Ser Asp 1145 1150 1155 Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu 1160 1165 1170 Gln Met Arg Asn Served Thr Gly Arg Thr Asp Val Asp Phe Leu 1175 1180 1185 Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg 1190 1195 1200 Asn Tyr Glu Only Gln Glu Asn Only Ile Leu Pro Lys Asn Only Asp 1205 1210 1215 Only Asn Gly Only Tyr Asn Ile Only Arg Lys Val Leu Trp Only Ile 1220 1225 1230 Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys 1235 1240 1245 Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser 1250 1255 1260 Val Lys His Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly 1265 1270 1275 Ser Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1280 1285 1290 <210> 36 <211> 3783 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 36 atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc ttcaaagctc 60 gagaaattca ccaactgtta ttcgttgagc aaaacactgc ggtttaaagc gattccagtc 120 ggcaagactc aagagaatat agacaataag cggctgttgg tggaagatga aaagcgcgcg 180 gaagactaca aaggggtgaa gaagttgttg gacagatact acctctcttt tatcaatgat 240 gtcttgcact caatcaaatt gaagaatctg aacaactaca tctccctctt cagaaagaaa 300 aaaaaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg hasaaaaaact aaaaaaaagg 360 atcgcgaaag cctttaaagg caacgaagga tacaaagtc tgttcagaa ggatataatt 420 gagacaattt tgccagagtt cctcgatgac aaggacgaga tgcgctggt caatcgttc 480 aacggattca cacaccatt cacaggctc tttgataatc gggaaaatat gttctctgag 540 gaggcaaagt ccactctat tgcgttcagg tgtatcaatg agaatctcac taggtacatt 600 tccacatgg atatctttga gaagttgac gcaatttg acagcacga agttcaggag 660 attaaggaga agatcctca ttccgatt gacgttgagg acttctcga gggtgagttt 720 tttaatttcg tgctcactca agagggtatc gacgtgtata atgcgatcat cggtgggttc 780 gtgactgagt ccggtgaaaa gattaaggga ttgaacgagt atcaacct ttacaaccaa 840 aagacgaaac agaagctgcc aaagttcaag cctctca aacaggttct ttcagaccgc 900 gagtcactct cgttctatgg ggaggctac acttcggatg aggagtcct ggaggtgttc 960 aggaatactc tcataagaa ttcggatt ttctctctta taaaaaaact ggaaaagttg 1020 tttaagaatt ttgacgaata ctctagcgcc ggcatatttg tgaaaaacgg cccggccata 1080 tcaacgataa gtaagatat cttcggcgaa tggaacgtga tcagagacaa atggaacgcg 1140 gagtatgacg attackcacct gagagaag gctgtcgtaa cggaagta cgaggatgat 1200 cgcaggaaaa gcttcaaaa gatcggaagt ttcagcctgg aacagttgca ggagtatgct 1260 gacgccgatc ttagcgtcgt cgagaagttg aaggaata tcatccaaaa ggtcgacgag 1320 atatataaag tctatggatc aagtgaaaaa ctgttcgacg ccgacttcgt tttggagaag 1380 tccctgaaga agaacgacgc tgttgttgcc attatgaagg atctgctcga cagcgtgaag 1440 agtttcgaga actatattaa ggctttttc ggggagggga aggagacta cagagatgag 1500 tccttctacg gagacttcgt cctcgcgtac gatatactcc tttaggtaga ccacatctac 1560 gacgcaatca gaaattacgt gacaaaag ccgtacagca aggacagtt cavacctac 1620 ttccagaacc cccagttcat gggcggctgg ngaaggaca aggaaacgga ttacaggggct 1680 acgatcctga ggtatggttc aaatactac ttggcgatta tggacaagaa gtacgccaag 1740 tgtctccaga agattgacaa agacgatgtc aatggcaatt atgagaagat caactacaag 1800 ctgcttccgg gtccgaacaa gatgctccca aaggttttct tcagcaagaa atggatggcc 1860 tactataacc caagcgagga catccagaag atttataaga acggtacgtt caagaagggc 1920 gacatgttca atcttaacga ctgtcacaag ctgatcgact tcttcaaaga ctcaattagc 1980 cggtacccaa agtggtctaa cgcctatgac ttcaactttt cggaaaccga gaagtacaag 2040 gatatagccg gattttatag agaggtggaa gagcagggct acaaggtgtc attcgagtcc 2100 gccagcaaga aggaagtgga caagctcgtg gaagagggta agctctacat gttccagatt 2160 tataataaag actttagcga taagagccac gggacaccta atctccacac aatgtatttc 2220 aagctgctct tcgacgagaa taaccacggc caaatcaggt tgtcaggagg ggctgaactc 2280 ttcatgcggc gcgctagcct taagaaggag gagcttgtag tccaccctgc gaatagtcca 2340 attgcgaata agaacccgga caatcctaaa aagactacaa cattgagcta cgacgtgtac 2400 aaggataaga ggttttccga ggatcagtac gagctccaca tcccgattgc gatcaacaag 2460 tgcccaaaga atatttcaa gataaacaca gaggtgcgtg tactcctgaa gcatgacgac 2520 aatccttacg tcattgggat tgatcggggc gagaggaacc tcctctatat tgtggtggtg 2580 gacgggaagg ggaacatagt cgaacagtac tcccttaacg aataattaa caatttcaac 2640 2700 gaggcgcggc aaaattggac aagtatcgag aacatcaagg aactcaaagc aggttatatc 2760 tctcaagttg tgcataagat atgcgagctg gttgagaagt atgacgcagt gatcgctctt 2820 gaggacctca actcgggctt taagaattct agagttaaag tggagaagca ggtctatcaa 2880 aagttcgaga agatgcttat agataagctc aactacatgg tcgataagaa atcgaaccca 2940 tgtgccaccg gcggcgcact caaaggttac aaataacaa acaaattcga gtccttcaaa 3000 tcgatgagta ctcagaatgg gttcatattt tatataccgg cgtggcttac gtctaagatc 3060 gacccgtcaa ctggttttgt caacctgttg aagacgaaat acacgtccat tgccgattca 3120 aaaaagttca tatctagttt tgatcgtatt atgtacgtcc your feet tcttttcgag 3180 tttgctctcg actacaaaaa cttttcgcgc accgatgcgg attacattaa aaaatggaaa 3240 ctctattcgt acggcaacag aatcaggatt tttcgcaacc ctaagaagaa taacgtcttt 3300 gattgggagg aagtttgctt gactagcgcg tacaaggagc tctttaataa gtatggcatt 3360 aactaccaac agggtgatat cagagcactg ctttgcgaac aatctgacaa ggctttctac 3420 tcatccttca tggctttgat gagcctgatg ctccagatga gaaattcaat tacaggcaga 3480 accgacgtgg atttcttgat ctccccggtt aaaaattctg atggcatctt ttacgatagc 3540 aggaactatg aagcgcaaga gaatgcgatt ctgccaaaaa atgcagacgc caacggtgcc 3600 tataacatcg ccaggaaagt cctgtgggcg atcggccagt tcaaaaaggc cgaagacgaa 3660 aaattggaca aggtcaaaat cgctatcagc aacaaagagt ggctggagta tgctcagaca 3720 tccgtaaagc ataagcgtcc tgctgccacc aaaaaggccg gacaggctaa gaaaaagaag 3780 tga 3783 <210> 37 <211> 1260 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 37 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Only Lys Ser Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Leu Ser Lys Thr 20 25 30 Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp 35 40 45 Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys 50 55 60 Gly Val Lys Lys Leu Asp Arg Tyr Tyr Leu Served Phe Ile Asn Asp 65 70 75 80 Val Leu His Ser Ile Lys Lys Asn Leu Asn Asn Tyr Ile Ser Leu 85 90 95 Phe Arg Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn 100 105 110 Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn 115 120 125 Glu Gly Tyr Lys Ser Leu Phe Lys Asp Ile Glu Thr Ile Leu 130 135 140 Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe 145 150 155 160 Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn 165 170 175 Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile 180 185 190 Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys 195 200 205 Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys 210 215 220 Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe 225 230 235 240 Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile 245 250 255 Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn 260 265 270 Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys 275 280 285 Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser 290 295 300 Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe 305 310 315 320 Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys 325 330 335 Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile 340 345 350 Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe 355 360 365 Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp 370 375 380 Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp 385 390 395 400 Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu 405 410 415 Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu 420 425 430 Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser 435 440 445 Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys 450 455 460 Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys 465 470 475 480 Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr 485,490,495 Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile 500 505 510 Leu Leu Lys Val Asp His With Tyr Asp With Arg Asn Tyr Val Thr 515,520,525 Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro 530 535 540 Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala 545 550 555 560 Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys 565,570,575 Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Val Asn Gly 580,585,590 Asn Tyr Glu Lys And Asn Tyr Lys Leu Pro Gly Pro Asn Lys Met 595,600,605 Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro 610 615 620 Serving Glu Asp With Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Gly 625 630 635 640 Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys 645,650,655 Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn 660,665,670 Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu 675,680,685 Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys 690,695,700 Glu Val Asp Lys Leu Val Glu Glu Gly Lys Tyr Met Phe Gln Ile 705 710 715 720 Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His 725 730 735 Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile 740,745,750 Arg Leu Serves Gly Gly Ala Glu Leu Phe Met Arg Arg Serves Lys 755,760,765 Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys 770,775,780 Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr 785 790 795 800 Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 805 810 815 Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val 820 825 830 Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp 835 840 845 Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly 850 855 860 Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn 865 870 875 880 Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu 885 890 895 Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile 900 905 910 Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys 915 920 925 Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn 930 935 940 Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln 945 950 955 960 Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys 965,970,975 Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile 980,985,990 Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Gln Asn Gly Phe 995 1000 1005 Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser 1010 1015 1020 Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala 1025 1030 1035 Asp Ser Lys Lys Phe Ile Ser Phe Asp Arg Ile Met Tyr Val 1040 1045 1050 Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe 1055 1060 1065 Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser 1070 1075 1080 Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn 1085 1090 1095 Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu 1100 1105 1110 Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg 1115 1120 1125 Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe 1130 1135 1140 Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr 1145 1150 1155 Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser 1160 1165 1170 Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn 1175 1180 1185 Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile 1190 1195 1200 Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu 1205 1210 1215 Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu 1220 1225 1230 Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro Ala 1235 1240 1245 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1250 1255 1260 <210> 38 <211> 3873 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 38 atgccgaaga agaagcgcaa ggtcgggggc gggggctcag gcgggggcgg gagcggcggc 60 gggggctctg ggggcggcgg cagcggcggg ggcggcagcg ggggcggcgg gtcgatgtca 120 aagctcgaga aattcaccaa ctgttattcg ttgagcaaaa cactgcggtt taaagcgatt 180 ccagtcggca agactcaaga gaatatagac aataagcggc tgttggtgga agatgaaaag 240 cgcgcggaag actacaaagg ggtgaagaag ttgttggaca gatactacct ctcttttatc 300 aatgatgtct tgcactcaat caaattgaag aatctgaaca actacatctc cctcttcaga 360 aagaaaacaa ggacagaaaa ggagaataag gaacttgaaa atttggagat caatctgagg 420 aaagagatcg cgaaagcctt taaaggcaac gaaggataca aaagtctgtt caagaaggat 480 ataattgaga caattttgcc agagttcctc gatgacaagg acgagattgc gctggtcaat 540 tcgttcaacg gattcacaac agcattcaca ggcttctttg ataatcgggga aaatatgttc 600 tctgaggagg caaagtccac ttctattgcg ttcaggtgta tcaatgagaa tctcactagg 660 tacatttcca acatggatat ctttgagaag gttgacgcaa tttttgacaa gcacgaagtt 720 caggagatta aggagaagat cctcaattcc gattatgacg ttgaggactt cttcgagggt 780 gagtttttta attcgtgct cactcaagag ggtatcgacg tgtataatgc gatcatcggt 840 gggttcgtga ctgagtccgg tgaaaagatt aagggattga acgagtatat caacctttac 900 aaccaaaaga cgaaacagaa gctgccaaag ttcaagcctc tttacaaaca ggttctttca 960 gaccgcgagt cactctcgtt ctatggggag ggctacactt cggatgagga agtcctggag 1020 gtgttcagga atactctcaa taagaattcg gagattttct cttctataaa aaaactggaa 1080 aagttgttta agaatttga cgaatactct agcgccggca tatttgtgaa aaacggcccg 1140 gccatatcaa cgataagtaa agatatcttc ggcgaatgga acgtgatcag agacaaatgg 1200 aacgcggagt atgacgatat tcacctgaag aagaaggctg tcgtaacgga gaagtacgag 1260 gatgatcgca ggaaaagctt caaaagatc ggaagtttca gcctggaaca gttgcaggag tatgctgacg ccgatcttag cgtcgtcgag aagttgaagg agataatcat ccaaaaggtc gacgagatat ataaagtcta tggatcaagt gaaaaactgt tcgacgccga cttcgttttg gagaagtccc tgaagaaga cgacgctgtt gttgccatta tgaaggatct gctcgacagc gtgaagagtt tcgagaacta tattaaggct tttttcgggg aggggaagga gactaacaga gatgagtcct tctacggaga cttcgtcctc gcgtacgata tactccttaa ggtagaccac atctacgacg caatcagaaa ttacgtgaca caaaagccgt acagcaagga caagttcaaa ctctacttcc aacccccca gttcatgggc ggctgggaca aggacaagga aacggattac 1740. agggctcga tcctgaggta tggttcaaaa tactacttgg cgattatgga caagaagtac gccaagtgtc tccagaagat tgacaaagac gatgtcaatg gcaattatga gaagatcaac tacaagctgc ttccgggtcc gaacaagatg ctcccaaagg ttttcttcag caagaaatgg atggcctact father cgaggacatc cagaagattt fathercgg tacgttcaag aagggcgaca tgttcaatct taacgactgt cacaagctga tcgacttctt caaagactca 2040 attagccggt acccaaagtg gtctaacgcc tatgacttca acttttcgga aaccgagaag 2100 tacaaggata tagccggatt ttatagagag gtggaagagc agggctacaa ggtgtcattc 2160 gagtccgcca gcaagaagga agtggacaag ctcgtggaag agggtaagct ctacatgttc 2220 cagatttata ataaagactt tagcgataag agccacgggga cacctaatct ccacacaatg 2280 tatttcaagc tgctcttcga cgagaataac cacggccaaa tcaggttgtc aggaggggct 2340 gaactcttca tgcggcgcgc tagccttaag aaggaggagc ttgtagtcca ccctgcgaat 2400 agtccaattg cgaataagaa cccggacaat ctaaaaaga ctacaacatt gagctacgac 2460 gtgtacaagg ataagaggtt ttccgaggat cagtacgagc tccacatccc gattgcgatc 2520 aacaagtgcc caaagaatat tttcaagata aacacagagg tgcgtgtact cctgaagcat 2580 gacgacaatc cttacgtcat tgggattgat cggggcgaga ggaacctcct ctatattgtg 2640 gtggtggacg ggaaggggaa catagtcgaa cagtactccc ttaacgaaat aattaacaat 2700 ttcaacggca tccgtatcaa gaccgactac cattcgttgc tggacagaa ggagaaggag 2760 agatttgagg cgcggcaaaa ttggacagt atcgacaaca tcaggact caagcaggt 2820 tatactctc aagttgtgca taagatatgc gagctggttg agagtatga cgcagtgatc 2880 gctcttgagg acctcaactc gggctttaag aattctagag ttaaagtgga gaagcaggtc 2940 tatcaaagt tcgagaat gcttatagat aagctcaact acatggtcga windowscg 3000 aaccatgtg ccaccggcgg cgcactcaa ggttaccaa taacaacaa attcgagtcc 3060 ttcaaatcga tgagtactca gatgggttc atatttta taccggcgtg gcttacgtct 3120 aagatcgacc cgtcaactgg tttgtcaac ctgttgaaga cgaatacac gtccattgcc 3180 gattcaaaa agttcatatc tagttttgat cgtattatgt acgtcccaga ggagatctt 3240 ttcgagtttg ctctcgacta caaaaacttt tcgcgcaccg atgcggatta cattaaaaaa 3300 tggaaactct attcgtacgg gcacaatc aggatttttc gcaacccta gagaatac 3360 gtctttgatt gggaggaagt ttgcttgact agcgcgtaca aggagctct tatagtat 3420 ggcattaact accaacaggg tgatatcaga gcactgcttt gcgaacaatc tgacaaggct 3480 ttctactcat ccttcatggc tttgatgagc ctgatgctcc agatgagaaa ttcaattaca 3540 ggcagaaccg acgtggattt cttgatctcc ccggttaaaa attctgatgg catcttttac 3600 gatagcagga actatgaagc gcaagagaat gcgattctgc caaaaaatgc agacgccaac 3660 ggtgcctata acatcgccag gaaagtcctg tgggcgatcg gccagttcaa aaaggccgaa 3720 gacgaaaaat tggacaaggt caaaatcgct atcagcaaca aagagtggct ggagtatgct 3780 cagacatccg taaagcatgg cagcccgaag aagaagcgca aggtgtccgg cggcagctcc 3840 ggcggcagcc cgaagaagaa gcgcaaagtg tga 3873 <210> 39 <211> 1290 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 39 Met Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Ser Met Ser Lys Leu Glu Lys Phe Thr Asn Cys 35 40 45 Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys 50 55 60 Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys 65 70 75 80 Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr 85 90 95 Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu 100 105 110 Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu 115 120 125 Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala 130 135 140 Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp 145 150 155 160 Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile 165 170 175 Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe 180 185 190 Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser Thr Ser 195 200 205 Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn 210 215 220 Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu Val 225 230 235 240 Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp 245 250 255 Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile 260 265 270 Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu 275 280 285 Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr 290 295 300 Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser 305 310 315 320 Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu 325 330 335 Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile 340 345 350 Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu 355 360 365 Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr 370 375 380 They contain Lys Asp and Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp 385 390 395 400 Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Ala Val Val Thr 405 410 415 Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Ile Gly Ser 420 425 430 Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Ser Val 435 440 445 Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr 450 455 460 Lys Val Tyr Gly Ser Ser Glu Lys Phe Asp Ala Asp Phe Val Leu 465 470 475 480 Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp 485,490,495 Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe 500 505 510 Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe 515 520 525 Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala 530 535 540 Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys 545 550 555 560 Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys 565 570 575 Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr 580 585 590 Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp 595 600 605 Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu 610 615 620 Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp 625 630 635 640 Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn 645 650 655 Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys 660 665 670 Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser 675 680 685 Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile 690 695 700 Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe 705 710 715 720 Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys 725 730 735 Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His 740 745 750 Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu 755 760 765 Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met 770 775 780 Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn 785 790 795 800 Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr 805 810 815 Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr 820 825 830 Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe 835 840 845 Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp Asn Pro 850 855 860 Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val 865 870 875 880 Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu 885 890 895 Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser 900 905 910 Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp 915 920 925 Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln 930 935 940 Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile 945 950 955 960 Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val 965 970 975 Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu 980,985,990 Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala 995 1000 1005 Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser 1010 1015 1020 Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu 1025 1030 1035 Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys 1040 1045 1050 Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser 1055 1060 1065 Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe 1070 1075 1080 Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile 1085 1090 1095 Lys Lys Trp Lys Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe 1100 1105 1110 Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys 1115 1120 1125 Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn 1130 1135 1140 Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp 1145 1150 1155 Light Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu 1160 1165 1170 Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu 1175 1180 1185 Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg 1190 1195 1200 Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp 1205 1210 1215 Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile 1220 1225 1230 Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys 1235 1240 1245 Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser 1250 1255 1260 Val Lys His Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly 1265 1270 1275 Ser Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1280 1285 1290 <210> 40 <211> 3852 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 40 atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tctgtttcaa 60 gattttacac atctgtaccc gctgagtaaa acagtgcggt tcgagctgaa acccatagga 120 aggaccctcg agcacatcca cgcgaagaat tttctgagcc aggatgaaac tatggctgat 180 atgtatcaaa aagttaaggt cattttggac gactatcatc gcgattttat tgccgacatg 240 atgggagagg tgaaactcac gaagcttgct gaattttacg acgtctatct gaagttcagg 300 aaaaatccta aggacgatgg gctgcaaaaa cagcttaaag accttcaagc tgtccttcgg 360 aaggaatcgg tgaagcctat agggtcaggt gggaagtaca aaacaggcta cgatagactc 420 tttggggcaa aactcttcaa agatggaaaa gagttgggtg acctcgcaaa attcgttata 480 gcccaagaag gtgagtcttc tccgaagctg gctcatcttg ctcattttga gaagttcagc 540 acgtatttta ctggatttca cgataatcgg aagaatatgt actcggatga agacaagcat 600 actgcaatag cgtacaggct catccatgag aatttgccga gattcatcga caatctgcaa 660 atcttgacaa caatcaaaca aaagcatagc gccctctatg atcagataat caacgagctc 720 acggcctccg ggctcgacgt ctccttggct tctcatcttg acgggtatca caagctcctt 780 acacaagagg ggatcacggc atacaacagg atcatagagg aggtgaatgg atatacaaat 840 aagcataacc agatatgcca caagagcgag cgcatagcga aacttagacc cttgcacaag 900 caaatccttt ctgacggaat gggagtgtca ttccttccgt ctaagttcgc ggatgatagt 960 gagatgtgcc aagcggtcaa cgaattttat cgcattata ctgacgtgtt cgcaaaggtg 1020 caaagtctct ttgacggatt tgatgatcac cagaaagacg ggatctatgt tgaacacaaa 1080 aaccttaatg aactgagcaa acaggcgttc ggcgactttg ctttgctggg gagggtcctt 1140 gatggatact acgtggacgt tgtcaatccg gagttcaatg agcggttcgc aaaggccaag 1200 actgacaatg cgaaagccaa gcttacaaaa gaaaaggaca aattcattaa aggagtccac 1260 tcactggctt ccctcgaaca agcaatagaa caccatacag ctagacacga cgatgagagt 1320 gttcaagccg gaaacttgg ccagtactc aacacggtt tggcggggt tgacaacccg 1380 attcagaaaa ttcacataa ccattcgacg attaaagggt ttctggaag ggaaggcct 1440 gctggggaac gggcgctccc gagatcaag tcaggaaaa acccagaat gacagctc 1500 agcagctga aggaactttt ggacaacgca tgaatgtgg cgcactcgc taagctgctg 1560 acactaaaa siaccttgga siaccaggat ggaattttt acggggatt tggggtgctt 1620 tacgacgagc tggctaaat tccaacctc tacaatagg ttagatta tctctcca 1680 aagccctttt ctaccgaaaa gtataagctc aacttcggca atccgaccct tctcaatggg 1740 tgggacctga tgggactt tgggactt tgggactt tggactt 1800 tacttggcg ttcttgataa ggctcataa aaagttttcg acaacgcccc taacactggt 1860 aagaacgtct accaaagat gtctacaa ctgttgcccg gccccaacaa atgcttcct 1920 aaagtgtttt tcgcaaatc gatctcgac tattaatc catctgccga gctccttgac 1980 aaatatgcta agggaccca taaaaagggt gataatttca acctgaagga ctgccacgcg 2040 cttatcgact tttcaaagc cgggataaat aagcatccgg agtggcaaca ttttggtttt 2100 aaattttcgc caacgtcgtc cttcgcgac ctttccgatt tctataggga agttgaacct 2160 caggggtacc aggtcaatt tgttgacatt aatgcggact acatgatga attggtggag 2220 caagggaagc tctacctt tcaatatat aaaagatt tctcgccaaa agcgcatggt 2280 aaaccgaatc ttcatacctt gtactttaaa gcacttttt cagaagataa cttggcggac 2340 ccgatctaca agctgaatgg ggaagctcag atcttctaca ggaagcttc gttggacatg 2400 aacgagacta ccatacatcg cgcgggagag gtgcttgaga acaaaatcc cgacaacccg 2460 aaaaagcggc aattcgttta cgacatcatc aagacaac ggtacacgca ggacaattt 2520 atgctccacg tcccattac catgaattttt ggagtccaag gcatgaccat taggaattc 2580 aaaaaagg tcaaccaag tattcagca tcatcag tcaatgtcat aggcatagat 2640 cggggagaaa ggcatctgtt gtatcttacc gtgatttact ctaagggtga atactggag 2700 caacggtcac ttaacgatat aaccacggcg tccgcgaacg gtacacaagt gaccactccc taccacaaaa tattggataa aagggagata gaacgcttga atgcccgcgt tggctggggt gagattgaga ccatcaaaga gcttaaatcg ggatatttgt ctcacgtcgt tcatcaaatt aaccaactca tgcttaagta caatgcaatc gttgtgctcg aggacctgaa ctttggtttc aaaagaggga ggttcaaggt ggaaaaacaa atttaccaga actttgaaaa cgcgcttatc aagaaattga atcaccttgt tttgaaagat aaggcagatg acgaaatcgg gtcgtataaa aatgcactcc agttgacaaa taatttcacg gatttgaagt cgatcggcaa gcaaacaggg ttcctctttt atgtgccagc gtggaataca tcaaaaattg atccggagac gggatttgtc 3180. gacttgctga agcctaggta tgagaacatt gcccaatctc aggccttttt cggcaaattc gataaaatat gctacaacac agacaaaggt tattttga ttcacattga ttacgccaaa tttacagata aggcgaaaaa cagcagacag aaatgggcta tctgttctca tggggacaaa cgctatgtct acgataagac ggctaatcaa aataaaggcg ccgcaaaagg fathergtg aatgatgagc tgaaaagctt gtttgcccgc taccatatca atgataaaca accaaacttg 3480 gtgatggaca tatgccagaa caatgacaaa gaattccaca agtcactcat gtgcctgctt 3540 aaaacccttt tggcgctgcg gtatagcaat gcatctagcg atgaagactt tattttgagt 3600 cccgtggcca acgacgaggg cgtgtttttt aattcagcct tggcggacga tacgcagccc 3660 cagaatgcgg acgcaaacgg cgcgtaccac attgcactga agggactgtg gcttctgaac 3720 gagctgaaaa atagcgacga cctgaataaa gtcaagttgg ccattgacaa tcaaacctgg 3780 ttgaatttcg ctcaaaatag aaagcgtcct gctgccacca aaaaggccgg acaggctaag 3840 aaaaagaagt ga 3852 <210> 41 <211> 1283 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 41 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Leu Phe Gln Asp Phe Thr His Leu Tyr Pro Leu Ser Lys Thr Val 20 25 30 Arg Phe Glu Leu Lys Pro Ile Gly Arg Thr Leu Glu His Ile His Ala 35 40 45 Lys Asn Phe Leu Ser Gln Asp Glu Thr Met Ala Asp Met Tyr Gln Lys 50 55 60 Val Lys Val Ile Leu Asp Asp Tyr His Arg Asp Phe Ile Ala Asp Met 65 70 75 80 Met Gly Glu Val Lys Leu Thr Lys Leu Ala Glu Phe Tyr Asp Val Tyr 85 90 95 Leu Lys Phe Arg Lys Asn Pro Lys Asp Asp Gly Leu Gln Lys Gln Leu 100 105 110 Lys Asp Leu Gln Ala Val Leu Arg Lys Glu Ser Val Lys Pro Ile Gly 115 120 125 Ser Gly Gly Lys Tyr Lys Thr Gly Tyr Asp Arg Leu Phe Gly Ala Lys 130 135 140 Leu Phe Lys Asp Gly Lys Glu Leu Gly Asp Leu Ala Lys Phe Val Ile 145 150 155 160 Ala Gln Glu Gly Glu Ser Ser Pro Lys Leu Ala His Leu Ala His Phe 165 170 175 Glu Lys Phe Ser Thr Tyr Phe Thr Gly Phe His Asp Asn Arg Lys Asn 180 185 190 Met Tyr Ser Asp Glu Asp Lys His Thr Ala Ile Ala Tyr Arg Leu Ile 195 200 205 His Glu Asn Leu Pro Arg Phe Ile Asp Asn Leu Gln Ile Leu Thr Thr 210 215 220 Ile Lys Gln Lys His Ser Ala Leu Tyr Asp Gln Ile Ile Asn Glu Leu 225 230 235 240 Thr Ala Ser Gly Leu Asp Val Ser Leu Ala Ser His Leu Asp Gly Tyr 245 250 255 His Lys Leu Leu Thr Gln Glu Gly Ile Thr Ala Tyr Asn Arg Ile Ile 260 265 270 Gly Glu Val Asn Gly Tyr Thr Asn Lys His Asn Gln Ile Cys His Lys 275 280 285 Ser Glu Arg Ile Ala Lys Leu Arg Pro Leu His Lys Gln Ile Leu Ser 290 295 300 Asp Gly Met Gly Val Ser Phe Leu Pro Ser Lys Phe Ala Asp Asp Ser 305 310 315 320 Glu Met Cys Gln Ala Val Asn Glu Phe Tyr Arg His Tyr Thr Asp Val 325 330 335 Phe Ala Lys Val Gln Ser Leu Phe Asp Gly Phe Asp Asp His Gln Lys 340 345 350 Asp Gly Ile Tyr Val Glu His Lys Asn Leu Asn Glu Leu Ser Lys Gln 355 360 365 Ala Phe Gly Asp Phe Ala Leu Leu Gly Arg Val Leu Asp Gly Tyr Tyr 370 375 380 Val Asp Val Val Asn Pro Glu Phe Asn Glu Arg Phe Ala Lys Ala Lys 385 390 395 400 Thr Asp Asn Ala Lys Ala Lys Leu Thr Lys Glu Lys Asp Lys Phe Ile 405 410 415 Lys Gly Val His Ser Leu Ala Ser Leu Glu Gln Ala Ile Glu His His 420 425 430 Thr Ala Arg His Asp Asp Glu Ser Val Gln Ala Gly Lys Leu Gly Gln 435 440 445 Tyr Phe Lys His Gly Leu Ala Gly Val Asp Asn Pro Ile Gln Lys Ile 450 455 460 His Asn Asn His Ser Thr Ile Lys Gly Phe Leu Glu Arg Glu Arg Pro 465 470 475 480 Ala Gly Glu Arg Ala Leu Pro Lys Ile Lys Ser Gly Lys Asn Pro Glu 485 490 495 Met Thr Gln Leu Arg Gln Leu Lys Glu Leu Leu Asp Asn Ala Leu Asn 500 505 510 Val Ala His Phe Ala Lys Leu Leu Thr Thr Lys Thr Thr Leu Asp Asn 515 520 525 Gln Asp Gly Asn Phe Tyr Gly Glu Phe Gly Val Leu Tyr Asp Glu Leu 530 535 540 Ala Lys Ile Pro Thr Leu Tyr Asn Lys Val Arg Asp Tyr Leu Ser Gln 545 550 555 560 Lys Pro Phe Ser Thr Glu Lys Tyr Lys Leu Asn Phe Gly Asn Pro Thr 565 570 575 Leu Leu Asn Gly Trp Asp Leu Asn Lys Glu Lys Asp Asn Phe Gly Val 580 585 590 Ile Leu Gln Lys Asp Gly Cys Tyr Tyr Leu Ala Leu Leu Asp Lys Ala 595 600 605 His Lys Lys Val Phe Asp Asn Ala Pro Asn Thr Gly Lys Asn Val Tyr 610 615 620 Gln Lys Met Val Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro 625 630 635 640 Lys Val Phe Phe Ala Lys Ser Asn Leu Asp Tyr Tyr Asn Pro Ser Ala 645 650 655 Glu Leu Leu Asp Lys Tyr Ala Lys Gly Thr His Lys Lys Gly Asp Asn 660 665 670 Phe Asn Leu Lys Asp Cys His Ala Leu Ile Asp Phe Phe Lys Ala Gly 675 680 685 Ile Asn Lys His Pro Glu Trp Gln His Phe Gly Phe Lys Phe Ser Pro 690 695 700 Thr Ser Ser Tyr Arg Asp Leu Ser Asp Phe Tyr Arg Glu Val Glu Pro 705 710 715 720 Gln Gly Tyr Gln Val Lys Phe Val Asp Ile Asn Ala Asp Tyr Ile Asp 725 730 735 Glu Leu Val Glu Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys 740 745 750 Asp Phe Ser Pro Lys Ala His Gly Lys Pro Asn Leu His Thr Leu Tyr 755 760 765 Phe Lys Ala Leu Phe Ser Glu Asp Asn Leu Ala Asp Pro Ile Tyr Lys 770 775 780 Leu Asn Gly Glu Ala Gln Ile Phe Tyr Arg Lys Ala Ser Leu Asp Met 785 790 795 800 Asn Glu Thr Thr Ile His Arg Ala Gly Glu Val Leu Glu Asn Lys Asn 805 810 815 Pro Asp Asn Pro Lys Lys Arg Gln Phe Val Tyr Asp Ile Ile Lys Asp 820 825 830 Lys Arg Tyr Thr Gln Asp Lys Phe Met Leu His Val Pro Ile Thr Met 835 840 845 Asn Phe Gly Val Gln Gly Met Thr Ile Lys Glu Phe Asn Lys Lys Val 850 855 860 Asn Gln Ser Ile Gln Gln Tyr Asp Glu Val Asn Val Ile Gly Ile Asp 865 870 875 880 Arg Gly Glu Arg His Leu Leu Tyr Leu Thr Val Ile Asn Ser Lys Gly 885 890 895 Glu Ile Leu Glu Gln Arg Ser Leu Asn Asp Ile Thr Thr Ala Ser Ala 900 905 910 Asn Gly Thr Gln Val Thr Thr Pro Tyr His Lys Ile Leu Asp Lys Arg 915 920 925 Glu Ile Glu Arg Leu Asn Ala Arg Val Gly Trp Gly Glu Ile Glu Thr 930 935 940 Ile Lys Glu Leu Lys Ser Gly Tyr Leu Ser His Val Val His Gln Ile 945 950 955 960 Asn Gln Leu Met Leu Lys Tyr Asn Ala Ile Val Val Leu Glu Asp Leu 965 970 975 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Ile Tyr 980,985,990 Gln Asn Phe Glu Asn Ala Leu Ile Lys Lys Leu Asn His Leu Val Leu 995 1000 1005 Lys Asp Lys Ala Asp Asp Glu Ile Gly Ser Tyr Lys Asn Ala Leu 1010 1015 1020 Gln Leu Thr Asn Asn Phe Thr Asp Leu Lys Ser Ile Gly Lys Gln 1025 1030 1035 Thr Gly Phe Leu Phe Tyr Val Pro Ala Trp Asn Thr Ser Lys Ile 1040 1045 1050 Asp Pro Glu Thr Gly Phe Val Asp Leu Leu Lys Pro Arg Tyr Glu 1055 1060 1065 Asn Ile Ala Gln Ser Gln Ala Phe Phe Gly Lys Phe Asp Lys Ile 1070 1075 1080 Cys Tyr Asn Thr Asp Lys Gly Tyr Phe Glu Phe His Ile Asp Tyr 1085 1090 1095 Ala Lys Phe Thr Asp Lys Ala Lys Asn Ser Arg Gln Lys Trp Ala 1100 1105 1110 How To Get His Gly Asp To Arg Tyr Val Tyr Asp To Thr Ala 1115 1120 1125 Asn Gln Asn Lys Gly Ala Ala Lys Gly Ile Asn Val Asn Asp Glu 1130 1135 1140 Leu Lys Ser Leu Phe Ala Arg Tyr His Ile Asn Asp Lys Gln Pro 1145 1150 1155 Asn Leu Val Met Asp Ile Cys Gln Asn Asp Lys Glu Phe His 1160 1165 1170 Lys Ser Leu Met Cys Leu Leu Lys Thr Leu Leu Ala Leu Arg Tyr 1175 1180 1185 Ser Asn Ala Ser Ser Asp Glu Asp Phe Ile Leu Ser Pro Val Ala 1190 1195 1200 Asn Asp Glu Gly Val Phe Phe Asn Ser Ala Leu Ala Asp Asp Thr 1205 1210 1215 Gln Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu 1220 1225 1230 Lys Gly Leu Trp Leu Leu Asn Glu Leu Lys Asn Ser Asp Asp Leu 1235 1240 1245 Asn Lys Val Lys Leu Ala Ile Asp Asn Gln Thr Trp Leu Asn Phe 1250 1255 1260 Ala Gln Asn Arg Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 1265 1270 1275 Ala Lys Lys Lys Lys 1280 <210> 42 <211> 3936 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 42 atgccgaaga agaagcgcaa ggtcgggggc gggggctcag gcgggggcgg gagcggcggc 60 gggggctctg ggggcggcgg cagcggcggg ggcggcagcg ggggcggcgg gtcgatgctg 120 ttccaggatt tcactcatct gtaccctctc tcaaagactg ttcggttcga gctcaagcct 180 attgggcgga ctctggagca catccacgcg aagaacttcc tcagccagga cgaaaccatg 240 gccgacatgt accagaaggt caaggtcatc ctcgacgact accacaggga cttcatcgcg 300 gacatgatgg gcgaggtgaa gctgacgaag ctcgccgagt tctacgacgt ctacctcaag 360 ttccgcaaga acccgaagga cgacggcctc cagaagcagc tcaaggacct gcaggccgtc 420 ctgaggaagg agtccgtcaa gcccatcggc agcggcggca agtacaagac cggctacgac 480 aggctgttcg gcgccaagct gttcaaggac ggcaaggagc tcggcgacct ggcgaagttc 540 gtgatcgcgc aggagggcga gagctccccc aagctggccc acctggccca cttcgagaag 600 ttcagcacgt acttcaccgg cttccacgac aacaggaga acatgtacag cgacgaggac aagcacacgg ccatcgccta ccgcctcatc cacgagaacc tgccccgctt catcgacaac ctgcagatcc tgacgaccat caagcagaag cactccgccc tgtacgacca gatcatcaac gagctcaccg cgagcggcct cgacgtgtcc ctcgccagcc acctcgacgg ctaccacaag 840 ctcctgaccc aggagggcat caccgcctac aaccgcatca tcggcgaggt gaacggctac accaacaagc acaaccagat ctgccacaag tccgagagga tcgccaagct caggcccctg cacaagcaga tcctcagcga cggcatgggc gtgagcttcc tcccgtccaa gttcgccgac gactccgaga tgtgccaggc cgtgaacgag ttctacaggc actacaccga cgtgttcgcc 1080 aaggtgcagt ccctgttcga cggcttcgac gaccaccaga aggacggcat ctacgtggag cacaagaacc tgaacgagct gtccaagcag gccttcggcg acttcgccct cctgggccgc gtgctggacg gctactacgt ggacgtcgtg aacccggagt tcaacgagcg cttcgcgaag gcgaagacgg acacgcgaa ggccaagctc accaaggaga aggacaagtt catcaagggc 1320 gtccacagcc tcgcgtccct ggagcaggcg atcgagcacc acacccgcg ccacgacgac 1380 gagtccgtgc aggccggcaa gctcggccag tacttcaagc acggcctggc cggcgtcgac 1440 aacccgatcc agagatcca siaaccac tccaccatca agggctctcct ggagagggag 1500 cgcccggcgg gcgagcgcgc gctccccaag atcaagtccg gcagaaccc cgagatgacg 1560 cagctcaggc agctgaagga gctgctcgac aacgcgctca acgtggcgca cttcgccaag 1620 ctgctcacga ccagaccac gctggacaac caggacggca acttctacgg cgagttcggc 1680 gtcctgtacg acgagctggc gagatccccg accctgtaca acaggtccg cgactacctg 1740 agccagaagc ccttctccac cgagaagtac aagctcact tcggcaaccc gaccctcctg 1800 aacggctggg acctcacaa ggaggaggac aacttcggcg tgatcctcca ggaggacggc 1860 tgctactacc tcgccctgct ggacaggcg cacaagaagg tctcgacaa cgccccgac 1920 accggcaaga acgtgtacca gagatggtg tacaagctgc tccccggcccc cacagatg 1980 ctgccgaagg tgttcttcgc gaagtccaac ctcgactact acaaccccag cgccgagctc 2040 ctggacaagt acgccaaggg cacgcacaag aagggcgaca acttcaacct caaggactgc 2100 cacgcgctga tcgacttctt caaggcgggc atcaacaagc accccgagtg gcagcacttc 2160 ggcttcaagt tcagcccgac ctccagctac agggacctca gcgacttcta ccgcgaggtg 2220 gagccccagg gctaccaggt gaagttcgtc gacatcaacg ccgactacat cgacgagctc 2280 gtcgagcagg gcaagctcta cctgttccag atctacaaca aggacttctc cccgaaggcc 2340 cacggcaagc cgaacctcca cacgctctac ttcaaggccc tcttcagcga ggacaacctg 2400 gccgacccga tctacaagct caacggcgag gcgcagatct tctaccgcaa ggccagcctg 2460 gacatgaacg aaacgaccat ccacagggcc ggcgaggtcc tggagaacaa gaacccggac 2520 aacccgaaga agaggcagtt cgtctacgac atcatcaagg acaagaggta cacccaggac 2580 aagttcatgc tccacgtccc gatcaccatg aacttcggcg tccagggcat gaccatcaag 2640 gagttcaaca agaaggtcaa ccagagcatc cagcagtacg acgaggtgaa cgtcatcggc 2700 atcgaccgcg gcgagaggca cctgctctac ctgacggtca tcaactccaa gggcgagatc 2760 ctcgagcagc gcagcctgaa cgacatcacg accgcgagcg ccaacggcac gcaggtcacc 2820 acgccgtacc acaagatcct cgacaagcgc gagatcgaga ggctgaacgc gcgcgtcggc 2880 tggggcgaga tcgaaacgat caaggagctc aagtccggct acctcagcca cgtcgtgcac 2940 cagatcaacc agctcatgct gagtcaac gcgatcgtgg tcctcgagga cctgaacttc 3000 ggcttcaaga ggggccgctt caaggtggag aagcagatct accagaactt cgagaacgcc 3060 3120 tacaagaacg cgctccagct gaccaacaac ttcacggacc tcaagtccat cggcaagcag 3180 acgggcttcc tgttctacgt gccggcgtgg aacacctcca agatcgaccc ggaaaccggc 3240 ttcgtcgacc tgctcaagcc gcgctacgag aacatcgcgc agtcccaggc gttcttcggc 3300 aagttcgaca agatctgcta caacaccgac aagggctact tcgagttcca catcgactac 3360 gcgaagttca ccgacaaggc caagaactcc aggcagaagt gggccatctg cagccacgg 3420 gacaagcgct acgtgtacga caagacggcg aaccagaaca agggcgcggc caagggcatc 3480 aacgtgaacg acgagctgaa gtccctcttc gcgcgctacc acatcaacga caagcagccg 3540 aacctcgtca tggacatctg ccagaacaac gacaaggagt tccacaagag cctgatgtgc 3600 ctgctcaaga ccctgctcgc cctccgctac tccaacgcga gctccgacga ggacttcatc 3660 ctcagccccg tcgcgaacga cgagggcgtg ttcttcaact ccgccctcgc ggacgacacg 3720 cagccgcaga acgccgacgc gaacggcgcc taccacatcg ccctcaaggg cctgtggctg 3780 ctcaacgagc tcaagaactc cgacgacctg aacaaggtga agctcgccat tgacaaccag 3840 acgtggctga atttcgctca gaataggccg aagaagaagc gcaaggtgtc cggcggcagc 3900 tccggcggca gcccgaagaa gaagcgcaaa gtgtga 3936 <210> 43 <211> 1311 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 43 Met Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Ser Met Leu Phe Gln Asp Phe Thr His Leu Tyr 35 40 45 Pro Leu Ser Lys Thr Val Arg Phe Glu Leu Lys Pro Ile Gly Arg Thr 50 55 60 Leu Glu His Ile His Ala Lys Asn Phe Leu Ser Gln Asp Glu Thr Met 65 70 75 80 Ala Asp Met Tyr Gln Lys Val Lys Val Ile Leu Asp Asp Tyr His Arg 85 90 95 Asp Phe Ile Ala Asp Met Met Gly Glu Val Lys Leu Thr Lys Leu Ala 100 105 110 Glu Phe Tyr Asp Val Tyr Leu Lys Phe Arg Lys Asn Pro Lys Asp Asp 115 120 125 Gly Leu Gln Lys Gln Leu Lys Asp Leu Gln Ala Val Leu Arg Lys Glu 130 135 140 Ser Val Lys Pro Ile Gly Ser Gly Gly Lys Tyr Lys Thr Gly Tyr Asp 145 150 155 160 Arg Leu Phe Gly Ala Lys Leu Phe Lys Asp Gly Lys Glu Leu Gly Asp 165 170 175 Leu Ala Lys Phe Val Ile Ala Gln Glu Gly Glu Ser Ser Pro Lys...

Claims

1. A fusion protein, wherein the fusion protein consists of SEQ ID NO: 81.

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