Infectious cDNA clone of EV71 strain and application thereof

Abstract

The present application belongs to the field of biological medicine, and provides a stable, infectious cDNA clone based on a clinically isolated EV71 strain, and its derivative clone containing various types of reporter genes, and various mutant clones constructed by taking the clone as the parent; and various recombinant viruses, subunit virus particles produced by using the clones; and animal models established by using the various recombinant viruses to infect animals; and the application of the viruses or subunit virus particles to vaccine development and diagnostic reagent; and the use of the virus as a gene therapy vector or an expression vector. The present application provides a new tool and approach for the detection, prevention and immunity of EV71 virus infection, and provides the possibility of using the infectious clone of the EV71 strain as a virus vector for gene therapy and vaccine development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the construction of an infectious cDNA clone based on a clinically isolated EV71 strain (js1), and the application of the cDNA clone and viruses derived therefrom, as well as viruses carrying a reporter gene and an established animal model in the development of antiviral drugs, vaccine development, and virus diagnosis. BACKGROUND

[0002] The prior art discloses that enterovirus is a general term for a class of viruses, including 3 types of poliovirus, 23 types of coxsackie virus A, 6 types of coxsackie virus B, 31 types of ECHO virus, and 68-71 types of enterovirus, totaling 67 types. After the traditional types, enterovirus discovered later is named according to the order of discovery, and now the newly discovered enterovirus includes 68, 69, 70, 71, and 72 types of enterovirus.

[0003] Research reports that EV71 virus is a single-stranded RNA virus, and its genome can encode a single long open reading frame (ORF), and two long non-coding regions 5'TURs and 3'TURs are contained in two segments of the genome. 5'TURs contain an internal ribosome entry site (IRES), which initiates the translation process of the virus (Hellen et al. Genes Dev. 2001; 15, 1593-1612). The open reading frame encoded by the virus is cut and processed into single viral proteins after translation, including structural proteins VP4, VP2, VP3, VP1 that constitute the virus particle, and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D responsible for virus replication (Racanillo, et al. Fields Virology. 2007, Fifth edition).

[0004] It has been reported that primates can be used as EV71 infection models. In 1978, Hashimoto et al. reported that 1.8-3.8 kg cynomolgus monkeys could be infected with an EV71 strain isolated from a 3-year-old child's fecal specimen after being isolated for 9 weeks. The EV71 virus was neurotoxic to the monkeys, and the monkeys showed clinical symptoms of neurological damage on the fourth day of infection, and the degree of damage was positively correlated with the viral titer. The EV71 virus could induce the monkeys to produce serum neutralizing antibodies (Hashimoto et al. Arch Virol. 1978; 56: 257-61). Zhang et al. used 3-3.5-year-old rhesus monkeys to establish an animal infection model showing symptoms of intracerebral infection, pulmonary edema, hemorrhage, and neurological damage. Intravenous and respiratory system infection can directly lead to neurological infection. Therefore, different infection routes can obtain models for different research purposes (Zhang et al. Lab Invest. 2011; 91: 1337-50). In addition, there is a rhesus monkey animal model that can cause central nervous system disease (Liu et al. Virology. 2011; 412: 91-100).

[0005] Non-primate animal models for EV71 have also been reported. For example, the mouse-adapted EV71 strain EV71 / MP4 can infect ICR mice, causing neurological and pulmonary damage (Chen et al. J Virol. 2007, 81 :8996-9003; Wang et al. J Virol. 2004, 78:7916-24). Arita et al. used immunodeficient non-obese diabetic severe combined immunodeficient mice (NOD / SCID mice) to obtain a mouse-adapted EV71 strain that can infect 3-week-old NOD / SCID mice. The NOD / SCID mouse model has suppressed natural killer cell function and lacks functional T and B cells. Moreover, the mouse-adapted strain mainly infects the central nervous system, heart, and skeletal muscle of the animals (Arita et al. J Virol. 2008, 82(4): 1787-97). Two-week-old or younger AG129 mice, which are immunodeficient mice deficient in interferon receptors alpha, beta, and gamma, can be infected with a natural EV71 strain, and exhibit symptoms of limb paralysis before the mice die (Khong et al. J Virol. 2012, 86(4):2121-31). Three-week-old transgenic mice expressing the EV71 receptor hSCARB2 can be successfully infected with the EV71 Isehara / Japan / 99 (Isehara) strain; studies have shown that constructing a mouse model of EV71 requires a special mouse-adapted strain or genetically deficient or modified mice.

[0006] It has also been reported that the genomic RNA of positive-strand RNA viruses is released and can be directly translated as an mRNA template after entering the cytoplasm of a host cell; the virus non-structural proteins produced by translation recruit the viral genome to form a replication complex to initiate viral gene replication and the life cycle, so the genomic RNA of a positive-strand RNA virus is infectious and can completely initiate the entire life cycle of the virus after being introduced into a host cell (Racaniello, et al. Science. 1981, 214(4523):916). The method for constructing an infectious clone generally uses total RNA from a virus-infected cell as a template for reverse transcription into complementary DNA (cDNA), and then clones the viral fragment into a cloning vector to form an infectious clone of the virus. The infectious clone is used to produce complete viral RNA by in vitro transcription, and then the viral RNA is transfected into a host cell to initiate the life cycle of the virus and produce progeny viruses. Alternatively, if the infectious clone carries a eukaryotic cell promoter, the plasmid can be directly transfected, and the full-length viral RNA is transcribed by the RNA polymerase of the host cell, thereby initiating the life cycle of the virus and producing progeny viruses.

[0007] Mouse model studies confirmed that the glutamic acid at position 145 of VP1 of EV71 is the main site of virus causing mouse death, and methylation of the lysine at position 149 of VP2 can synergize with the ability of VP1 145E to cause mouse death (Huang et al. Virology. 2012, 422(1): 132-43). This virus site is prone to mutation to 145G during in vitro passage of the virus, resulting in a decrease in the ability of the virus to infect animals (Yi et al. Unpublished data).

[0008] Based on the basis and status of the prior art, the inventors of the present application propose to provide an infectious cDNA clone of an EV71 strain and applications thereof. SUMMARY

[0009] The purpose of the present application is to provide an infectious cDNA clone of an EV71 strain and applications thereof based on the basis and status of the prior art. Specifically, it relates to a stable infectious cDNA clone of an EV71 strain, which can replicate itself, produce progeny virus particles, and express a reporter gene in cells.

[0010] Another technical problem to be solved by the present application is to provide recombinant viruses or subunit virus particles, plasmids, etc. constructed based on the above-mentioned clone, to provide support for the construction of animal models, vaccine development, and the development of antiviral drugs.

[0011] The present application isolates an EV71 strain (named js1) from the clinic, which does not require mouse adaptation mutations and can infect mice without genetic background changes. By constructing its infectious clone, virus particles with stable genetic sequences can be produced, which can infect ordinary mice and establish a simple and efficient EV71 animal infection model.

[0012] More specifically, the present application provides a cDNA comprising the nucleic acid sequence of an EV71 strain and the nucleic acid sequence of a low-copy plasmid backbone; wherein the nucleic acid sequence of the EV71 strain covers the 5' to 3' positive sequence of the EV71 virus, including the 5' and 3' non-coding regions and an open reading frame encoding a viral protein.

[0013] Preferably, it further comprises the sequence of a reporter gene luciferase or fluorescent protein inserted into the nucleic acid sequence of the EV71 strain.

[0014] The amino acid sequence of the viral protein open reading frame is shown in SEQ ID NO 4.

[0015] The coding sequence of the low-copy plasmid backbone is shown in SEQ ID NO 3.

[0016] The nucleic acid sequence of the EV71 strain is shown as SEQ ID NO 2.

[0017] In a preferred embodiment of the present application, the infectious cDNA clone of the EV71 strain has the sequence shown as SEQ ID NO 1.

[0018] In an embodiment of the present application, a stable infectious cDNA clone of a clinical isolate of EV71 strain (nucleic acid sequence 1) and its derivative clones containing various reporter genes (nucleic acid sequence 5, nucleic acid sequence 6, and nucleic acid sequence 7) are provided.

[0019] 6), and various mutant clones constructed using the above as the parent. The viral RNA produced by these clones can self-replicate in cells, produce progeny viral particles, and express the reporter genes.

[0020] The present application also includes recombinant viral clones containing heterologous reporter sequences or target genes constructed using the sequences shown in nucleic acid sequence 6 or nucleic acid sequence 7 as the parent, and the sequences thereof.

[0021] The present application also includes various chimeric viral infectious clones and recombinant viral clones containing heterologous reporter sequences or target genes, and various chimeric viruses and viral particles containing reporter genes or exogenous genes produced by the above.

[0022] The present application also includes recombinant viral clones in which heterologous resistance sequences are inserted into the open reading frame of viral proteins constructed using the full-length infectious clone sequence, and the sequences thereof.

[0023] In particular, the present application provides an infectious cDNA clone (nucleic acid sequence 1) of a clinically isolated EV71 strain (js1), which comprises a full-length nucleic acid sequence of the EV71 strain (js1) (nucleic acid sequence 2) and a low-copy plasmid backbone (nucleic acid sequence 3). The nucleic acid sequence 2 encompasses the 5' to 3' positive-sense sequence of the EV71 virus, which includes the viral 5' and 3' non-coding regions and an open reading frame encoding viral proteins. The open reading frame viral encoded proteins (protein sequence 4) are inserted with a reporter gene, luciferase NanoLuc (Nluc) and fluorescent protein EGFP in the infectious clone (nucleic acid sequence 1), respectively, to form an infectious clone with Nluc (nucleic acid sequence 5) and an infectious clone with EGFP (nucleic acid sequence 6), and derivatives (derivative) such as subgenomic replicon including deletion of structural proteins, adapted virus, live-attenuated virus, defective virus and replication-competent non-infectious virus, etc. obtained by means of changing nucleic acid based on these clones.

[0024]

[0025]

[0026] Nucleic acid sequence 3, SEQ ID NO 3: AGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGT GGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGA CTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCA GGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATC ACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCT CCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCC ACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACC GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACT GGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACT GCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCG GTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGA CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTAT CAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTA CTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGC.

[0027] Protein sequence 4, SEQ ID NO 4: MGSQVSTQRSGSYENSNSATEGSTINYTTINYYKDSYAATAGKQSLKQDPDKFANPVKDIFTEMAAPLKS PS AEACGYSDRVAQLTIGNSTITTQEAANIIVGYGEWPSYCSDSDATAVDKPTRPDVSVNRFYTLDTKLWEKSSKG WYWKFPDVLTETGVFGQNAQFHYLYRSGFCIHVQCNASKFHQGALLVAVLPEYVIGTVAGGTGTEDTHPPYKQTQPG ADGFELQHPYVLDAGIPISQLTVCPHQWINLRTNNCATIIVPYINALPFDSALNHCNFGLLVVPISPLDYDQGATPV IPITITLAPMCSEFAGLRQAVTQGFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILE VNNVPTNATSLMERLRFPVSAQAGKGELCAVFRADPGRNGPWQSTLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKM LIAYTPPGGPLPKDRATAMLGTHVIWDFGLQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGA PNTAYIIALAAAQKNFTMKLCKDASDILQTGTIQGDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGK VPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLEGTTNPNGYANWDIDITGYAQMR RKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSP ASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFK ANPNYAGNSIKPTGASRTAITTLGKFGQQSGAIYVGNFRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCDCQTGVYYCNSMRKHYPVSFSKPSLIYVEASEYYPARYQSHLMLAQGHSEPGDCGGILRCQHGVIGIVSTGGNGL VGFADVRDLLWLDEEAMEQGVSDYIKGLGDAFGTGFTDAVSREVEALKNYLIGSEGAVEKILKNLIKLISALVIVIR SDYDMVTLTATLALIGCHGSPWAWIKAKTASILGIPIAQKQSASWLKKFNDMANAAKGLEWVSNKISKFIDWLKEKI VPAAREKVEFLNNLKQLPLLENQISNLEQSAASQEDLEVMFGNVSYLAHFCRKFQPLYATEAKRVYALEKRMNNYMQ FKSKHRIEPVCLIIRGSPGTGKSLATGIIARAIADKYHSSVYSLPPDPDHFDGYKQQVVTVMDDLCQNPDGKDMSLF CQMVSTVDFIPPMASLEEKGVSFTSKFVIASTNASNIIVPTVSDSDAIRRRFYMDCDIEVTDSYKTDLGRLDAGRAA KLCSENNTANFKRCSPLVCGKAIQLRDRKSKVRYSVDTVVSELIREYSNRSAIGNTIEALFQGPPKFRPIRISLEEK PAPDAISDLLASVDSEEVRQYCRDQGWIIPEAPTNVERHLNRAVLVMQSITTVVAVVSLVYVIYKLFAGFQGAYSGA PKQVLKKPALRTATVQGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVE LVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMY NFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQGEIQWVKPNKETGRLNINGPTRTKLEPSVFHD IFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTLHEPDEYIKEAALHYANQLKQLEINTSQMSMEEACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSRMKFYMDKYGLDLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYEAFHANPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYSEEAISLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRALLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSPCFNEVNWGNATFLKRGFLPDEQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPNYENLRRNWLELF.

[0028]

[0029]

[0030] The present application also includes the expression products of these cDNA clones.

[0031] The present application also includes double stranded DNA containing the above cDNA, double stranded DNA capable of producing full-length infectious clone sequences, positive-sense cDNA or negative-sense cDNA.

[0032] The present application also includes plasmids containing the above cDNA or double stranded DNA.

[0033] Preferably, the plasmid is capable of transcribing full-length infectious RNA of EV71 strain or its mutants, capable of producing plasmids containing full-length infectious RNA of EV71 strain (jsl) by in vitro transcription, capable of producing plasmids containing full-length infectious RNA of EV71 strain (jsl) by in vitro transcription and derivative plasmids.

[0034] The derivative plasmids include:

[0035] A. recombinant virus clones obtained by replacing part of the sequences of the full-length infectious clone of EV71 strain (jsl) in claim 1 with part of the sequences of other isolates;

[0036] B. mutant virus clones obtained by mutating the sequences in the full-length infectious clone of EV71 strain (jsl) in claim 1 or 2 by genetic mutation;

[0037] C. derivative clones such as live-attenuated, replication competent non-infectious and defective variants of viruses produced from the full-length infectious clone of EV71 strain (jsl) by adaptive mutation.

[0038] The present application provides a plasmid containing the above double stranded DNA or its derivatives.

[0039] Preferably, it is capable of transcribing full-length infectious RNA of EV71 strain or its mutants.

[0040] The present application also provides a vaccine or viral vector prepared according to the above plasmid.

[0041] The present application provides a viral particle prepared from the above cDNA clone or plasmid.

[0042] For example, live-attenuated virus particles, replication competent non-infectious virus particles and defective variants of virus particles;

[0043] The virus can be used to isolate, purify and obtain anti-EV71 virus antibodies by immunizing animals, and can be used to screen human antibody libraries, or to prepare kits for detecting EV71 virus and various cell lines, tissues and animal infection models.

[0044] The cell lines, tissues and animal infection models can be used to screen anti-EV71 virus drugs.

[0045] The present application also includes a method for detecting and preparing the virus vector and virus particles.

[0046] For example, the virus particles are used to immunize animals and isolate antibodies, or to screen human antibody libraries.

[0047] In another aspect, the present application provides a kit for detecting EV71, which contains the cDNA or virus particles.

[0048] The present application also provides a method for preparing anti-virus EV71 drugs, which uses the cDNA or virus particles to construct cell or animal models for screening anti-virus EV71 drugs.

[0049] The infectious clone (nucleic acid sequence 1) of the present application is a complete plasmid composed of DNA sequences, which contains a full-length EV71 strain (js1) nucleic acid sequence (nucleic acid sequence 2) and a low-copy plasmid backbone sequence (nucleic acid sequence 3). The plasmid is a closed double-stranded DNA covalently bonded, which contains a positive-sense strand identical to the mRNA sequence and a negative-sense strand complementary thereto.

[0050] The full-length nucleic acid sequence of EV71 strain (jsl) contained in the infectious clone of the present application (nucleic acid sequence 1) includes a non-translated region (NTR) at the 5' end of the positive sense sequence, an open reading frame (ORF) and a 3'-NTR at the 3' end of the viral full-length nucleic acid sequence (nucleic acid sequence 2).

[0051] In the infectious clone, the 5' end of the viral full-length nucleic acid sequence contains a T7 promoter (TAA TAC GACTC ACTATAG G, SEQ ID NO 7) Figure 1 A), which can be used to transcribe the viral full-length RNA in vitro by using a commercial T7 transcription kit; and the 3' end of the viral full-length nucleic acid sequence contains a 30-nucleotide long polyA tail (AAAAA AAAAAAAAAA AAAAA AAAAA AAAAA, SEQ ID NO 8) Figure 1 A).

[0052] The clinical isolated strain was used to infect RD cells, and total RNA was extracted from the cells when cytopathic effect appeared. The extracted total RNA was subjected to reverse transcription using EV71 specific primers (GCT AG CGCT tt tttttttt tttttttt ttttttt ttttt, SEQ ID NO 9), and then the cDNA obtained by reverse transcription (Superscript II reverse transcriptase) was subjected to PCR amplification. The full-length EV71 genome was divided into four segments for amplification Figure 1 A), and the amplification primers were F1 (S: GAC GC GGCCG C TAA TAC GACTC ACTATAG GTTAAAACAGC CTGT GGGT TGCAC CC, SEQ ID NO 10; As: GCACTG CACGT GGATGC AGAAC, SEQ ID NO 11), F2 (S: GAC GCG GCCGC G TTCT GCAT CCAC GTGCA GTGC, SEQ ID NO 12; As: AAGTC GCGAGAGCT GTCTTC CC, SEQ ID NO 13), F3 (S: GAC GCG GCCGCGGGAA GACAG CTCTCG CGACTT, SEQ ID NO 14; As: AATTG TACAT CATG GTGC GATGG GTAGG, SEQ ID NO 15), F4 (S: GAC GC GGCCGC CCTAC CCATCG CACCATG ATGTAC AATT, SEQ ID NO 16; As: GCT AGC GCT tttttttttttttttt tttttttt ttttttGCT ATTCT GGTTAT AACAA ATTTA CCCCCA CCAG, SEQ ID NO17), the amplified fragment was cloned into pANCR vector by stepwise cloning, to obtain the final full-length cDNA clone, named pEV71-js1 Figure 1 A), after linearization by HindIII in vitro, the infectious clone was transcribed into full-length viral RNA and polyA tail at the 3' end by T7 transcription kit. After the in vitro generated viral RNA was introduced into host cells such as Vero cells by electroporation or transfection, the viral RNA served as a translation template to translate the ORF, to produce viral polypeptides (protein sequence 4); the viral polypeptides were processed to form viral structural proteins and non-structural proteins, to initiate the entire viral life cycle and produce progeny viruses.

[0053] Due to the coding-based mergence, the same functional protein product can be obtained by changing codons without changing the protein sequence; the present application includes other nucleic acid sequences encoding the same as "protein sequence 4" and infectious clones.

[0054] The virus produced by the infectious clone (nucleic acid sequence 1) of the present application exhibits strong replication ability in cells Figure 2 ), and can be used to infect in vitro cultured cell lines, neural tissues, mice Figure 6 or monkeys, etc. to establish viral infection cell models and animal infection models, for drug development.

[0055] By modifying the infectious clone (nucleic acid sequence 1), a reporter gene is inserted into a specific region of the virus (before the VP4 protein coding region), and the reporter gene is translated by the IRES initiation of the virus. Additional amino acid sites (AITTL) are added at the C-terminus of the reporter gene Figure 1B), which can be recognized and cleaved by the 3C protease of the virus, resulting in a normal N-terminus of VP4. We successfully inserted the reporter genes luciferase NanoLuc (Nluc) and fluorescent protein EGFP into this infectious clone (nucleic acid sequence 1), resulting in the infectious clone with Nluc (nucleic acid sequence 5) and the infectious clone with EGFP (nucleic acid sequence 6) Figure 1 B). Each reporter gene was linked to the pEV71-js1 plasmid (nucleic acid sequence 1) by fusion PCR, resulting in pEV71-js1-Nluc (nucleic acid sequence 5) Figure 1 B) and pEV71-js1-EGFP (nucleic acid sequence 6) Figure 1 B); this infectious clone was linearized by HindIII in vitro, and the full-length viral RNA was transcribed by a T7 transcription kit. The in vitro transcribed viral RNA was introduced into host cells such as Vero cells by electroporation or transfection, and the viral life cycle was initiated to produce progeny viruses Figure 2 ). The virus expresses the reporter genes Nluc and EGFP during replication. Nluc can be detected using a commercial luciferase activity detection kit Figure 5 ). The expression of EGFP can be observed using a fluorescence microscope Figure 4 ) or detected using a flow cytometer. The progeny viruses containing the reporter gene fragment re-infect new cells and can effectively replicate in the new cells. The expression level of the reporter gene, which is in the same open reading frame as the viral protein, reflects the level of viral protein and also reflects the level of viral replication. The recombinant virus containing the reporter gene does not lose the reporter gene during continuous passage for a long period of time Figure 4 ). Using this recombinant virus containing the reporter gene, the viral replication and packaging level can be quickly and conveniently detected, and it can be used for the study of the viral life cycle, virus-host interaction, virus immunology, and the development of antiviral drugs, etc.

[0056] This infectious clone (nucleotide sequence 1) was modified, similar to other enteroviruses, by deleting the VP4-VP3-VP2-VP1 region of the viral structural proteins. This resulted in a subgenomic replicon, a replication-competent non-infectious virus. This subgenomic replicon is capable of viral gene replication but lacks the viral structural proteins, preventing the production of progeny viruses. Furthermore, the subgenomic replicon RNA can be trans-complemented by expressed structural proteins to form recombinant subviral particles (RSPs) (Barclay, et al. J Gen Virol. 1998, 79:1725-1734; Jia, et al. J Virol. 1998, 72:7972-7977). These subviral particles are capable of one round of infection, but because the genome lacks the encoded structural proteins, they are unable to be packaged into further viral particles after infection, resulting in defective virus variants. These non-replicating virus particles can serve as a form of vaccine.

[0057] This infectious clone (nucleotide sequence 1) can be modified to produce a live-attenuated virus, which can be used as a vaccine. Referring to the attenuation strategy of Polio virus, also in the Picornaviridae family, mutations in the 5'NTR can be used to construct an attenuated vaccine (Arita, et al. J Virol. 2008, 82: 1787-1797).

[0058] The virus produced by the infectious clone infects mice to establish a convenient and stable animal infection model. In this application, it was found that the mortality rate of the initially isolated strain after more than three passages after infecting newborn mice decreased. Sequencing revealed that the VP1 145 position of the passaged virus had a mutation from E to G. Therefore, the consistency of the mouse model established by infection with the isolated virus is poor. The use of the infectious clone to produce the virus can ensure that the viral sequence is not affected by cell passage. The virus obtained by the above infectious clone was used to infect newborn mice of different strains (ICR, Balb / c, C57), and 100% mortality was obtained within 9 days ( Figure 6 B), but the virus carrying the 145G mutation of VP1 did not cause death in mice after infection ( Figure 6 C), this animal model can be conveniently used for the evaluation of antiviral drugs and vaccines, etc.

[0059] The new enterovirus type 71 (EV71) belongs to the Picornaviridae family ( picornaviridae ) is a member of the enterovirus group. EV71 is the main pathogen that causes hand, foot and mouth disease in children worldwide. It can cause mild and severe hand, foot and mouth disease in children. The virus can infect the central nervous system and cause central nervous system damage, but its mechanism is unclear. There is currently no effective antiviral drug for the treatment of EV71. The present invention isolates a clinical EV71 strain and constructs a stable full-length cDNA clone of the virus by molecular cloning. By in vitro transcription of RNA and transfection of Vero cells, it is confirmed that the viral RNA derived from the cDNA clone can produce EV71 virus; further, the present application constructs a recombinant virus containing the reporter genes Gluc (Gaussia luciferase) and EGFP, and confirms that the recombinant virus containing the reporter genes Gluc and EGFP has the ability to infect host cells and cause cytopathic effects. The EV71 virus derived from the infectious clone is used to infect immune-competent ICR, Bab / C and C57 suckling mice, resulting in 100% of the infected mice showing symptoms of neurological damage and death within 10 days.

[0060] The present invention provides a stable infectious cDNA clone based on a clinically isolated EV71 strain and its derivative clones containing various reporter genes, as well as various mutant clones constructed using the clones as the parent; various recombinant viruses and subunit virus particles produced using these clones; and animal models established by infecting animals with the various recombinant viruses produced by these clones; as well as the use of these viruses or subunit virus particles in vaccine development and diagnostic reagents; and the use of these viruses as gene therapy vectors or expression vectors.

[0061] The present invention has the following advantages: the present invention includes various recombinant viruses and subunit virus particle plasmids constructed by molecular biology using these cloned plasmids as mothers.

[0062] The present invention also includes various recombinant viruses and subunit virus particles that can be produced using these clones; they contain the above-mentioned cDNA.

[0063] The present invention also includes animal infection models constructed using various recombinant viruses that can be produced by these clones.

[0064] The present invention also includes the use of these viruses or subunit virus particles and animal models for the development of vaccines and diagnostic reagents.

[0065] The present invention also includes animal models established using these viruses or subunit virus particles for the development of vaccines and antiviral drugs.

[0066] The present application also includes the use of the virus or subvirus unit plasmid as a gene therapy vector or expression vector plasmid and the use of the virus or subvirus particles produced by the plasmid.

[0067] The present application provides a new tool and approach for the detection, prevention and immunization of EV71 virus infection and provides the possibility of using the infectious clone of the EV71 strain as a viral vector for gene therapy and vaccine development. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 Construction of the infectious cDNA clone of EV71 strain js1, wherein (A) the strategy of infectious clone construction; the full genome of Zika virus model, the black column at both ends represents the 5'-NTR and 3'-NTR respectively; the region of viral structural protein and non-structural protein is shown in the figure; the full-length sequence of the virus is divided into four segments for amplification, wherein the first segment F1 contains a T7 sequence, and the fourth segment F4 contains a polyA30 sequence introduced by PCR primers; the synthesized sequence is sequentially connected into the pACNR vector as shown in the figure to obtain the full-length clone; (B) through fusion PCR, the Nluc or EGFP gene is fused in-frame at the N-terminus of VP4, and additional amino acid sequence AITTL is added at the C-terminus of the Nluc or EGFP gene, so as to be cut by viral protease to produce the correct N-terminus of VP4.

[0069] Figure 2 Replication and infection ability of the virus produced by the infectious cDNA clone of EV71 strain js1, wherein the infectious clone plasmid is used as a template to transcribe into viral RNA in vitro, the viral RNA is electrically introduced into Vero cells, the supernatant virus is collected, and the titer is titrated by plaque assay on Vero cells; the plaque of the infectious clone (Clone-WT) is compared with the plaque of the parent virus (Parent) as shown in the figure (top), and the virus produced by the infectious clone at the same titer is used to infect Vero cells again (MOI=0.1), the cell supernatant collected at different time (h.p.i) after infection is titrated by plaque assay, and the growth curves of the two are shown in the figure (bottom), and the viral titer is represented by PFU / ml.

[0070] Figure 3: Generation of recombinant virus containing reporter Nluc and EGFP, wherein (A) infectious clone plasmid containing reporter Nluc and EGFP, infectious clone plasmid without reporter, were transcribed into viral RNA in vitro, viral RNA was introduced into Vero cells by electroporation, supernatant virus was collected, and titer titration was performed on Vero cells by plaque assay, and the plaques generated by recombinant virus containing each reporter were compared with the plaques generated by virus without reporter; (B) Vero cells were infected again with virus containing each reporter and virus without reporter at the same titer (MOI = 0.1), supernatant was collected at different days after infection, and growth curves were obtained by plaque assay; viral titer was expressed by PFU / ml.

[0071] Figure 4 : Stability of recombinant virus containing EGFP reporter, wherein the supernatant of cells infected with recombinant virus EV71-EGFP was diluted 1:10 to re-infect new Vero cells, and after two days of infection, the cells were observed under a fluorescence microscope and the supernatant was collected, and the new Vero cells were re-infected again with a dilution of 1:10 (C+1), and after two days of infection, the cells were observed under a fluorescence microscope and the supernatant was collected for re-infection; the cells were sequentially infected, and the expression of EGFP in the infected cells was observed.

[0072] Figure 5 : Generation of recombinant virus containing Nluc reporter and activity of Nluc, wherein infectious clone plasmid containing reporter Nluc and plasmid containing VP1 E145G and 3C C147A mutation were transcribed into viral RNA in vitro, viral RNA was introduced into Vero cells by electroporation, and cells were collected at different time points after electroporation, and the activity of Nluc in the cells was detected, and C147A is a deletion mutation of 3C protease activity.

[0073] Figure 6 : Generation of virus by infectious cDNA clone of EV71 strain js1 and construction of animal infection model by infecting mice, wherein (A) virus generated by infectious cDNA clone was used to infect different strains of 3-day-old fetal mice (1.4 x 104 pfu per mouse), and the mice were observed 5 days after infection; (B) survival curve of mice after virus infection (n = 5 / group); (C) virus generated by infectious cDNA clone (WT) and clone carrying VP1 E145G mutation was used to infect 3-day-old ICR mice, and the survival curve of mice (n = 5 / group) was obtained. DETAILED DESCRIPTION

[0074] The methods used in the present application are all conventional molecular biology methods, and specific operation details are not described again.

[0075] Example 1: Construction of an infectious cDNA clone of EV71 strain js1.

[0076] like Figure 1 As shown in A, the virus isolated from the stool specimen was cultured in RD cells. When the cells showed obvious cytopathic effects, total cellular RNA was extracted and reverse transcriptase was used by Superscript II (Invitrogen) with sequence-specific primers (GCT AG CGCT The cDNA was used as a template for reverse transcription, and PCR amplification was performed in four sections using the high-fidelity enzyme Super Fi (Invitrogen). The amplification primers were F1 (S: GAC GC GGCCG C TAA TAC GACTC ACTATAG GTTAAA ACAGC CTGT GGGT TGCAC CC; As:GCACTG CACGT GGATGCAGAAC), F2(S:GAC GCG GCCGC G TTCT GCAT CCAC GTGCA GTGC; As:AAGTC GCGA GAGCTGTCTTC CC), F3(S:GAC GCG GCCGC G GGAA GACAG CTCTCG CGACTT; As:AATTG TACAT CATGGTGC GATGG GTAGG), F4 (S: GAC GC GGCCGC CCTAC CCATCG CACCATG ATGTAC AATT; As: GCT AGC GCT The amplified F4 fragment was first digested with restriction enzymes NotI / AfeI and then ligated with the pANCR vector digested with the same restriction enzymes to obtain the pANCR-F4 plasmid. The PCR-amplified F3 fragment was ligated into pANCR-F4 using NruI / BsrGI to obtain the pANCR-F34 plasmid. The PCR-amplified F2 fragment was ligated into pANCR-F34 using PmlI / NruI to obtain pANCR-F234. The PCR-amplified F1 fragment was ligated into pANCR-F234 using NotI / PmlI to obtain the final full-length cDNA clone, which was named pEV71-js1.

[0077] To construct infectious clone plasmid with reporter gene EGFP (such as Figure 1 B), three sequences were fused by fusion PCR, in which EGFP-F1 is EV71 5UTR sequence, PCR amplification primer is S: CCTGACGTGTGCAGCGGG, SEQ ID NO 18, As: cctcgcccttgctcacCATcatatgGTTTAGCTGTGTTAAGGGTCAAGA, SEQ ID NO 19, EGFP-F2 is a fragment containing EGFP, PCR amplification primer is S: TCTTGACCC TTAACACAGCTAAACcatatgATGgtgagcaagggcgagg, SEQ ID NO 20, As: CGCTGTGTAGACACTTGCGAACCAAGAGTGGTGATCGCatgcatcttgtacagctcgtccatgccg, SEQ ID NO 21, EGFP-F3 is a fragment containing VP4 and VP2 regions, PCR amplification primer is S: cggcatggacgagctgtacaagatgcATGCGATCACCACTCTTGGTTCGCAAGTGTCTACAGCG, SEQ ID NO 22; As: CTGCA CGTGGATGCA GAACCC, SEQ ID NO 23, after the three fragments were fused by fusion PCR, they were ligated into pEV71-js1 plasmid by NotI / PmlI, replacing the sequence in the original plasmid, to obtain pEV71-js1-EGFP plasmid.

[0078] To construct infectious clone plasmid with reporter gene Nluc (such as Figure 1 B), two sequences were fused by fusion PCR, in which Nluc-F1 is EV71 5UTR sequence, PCR amplification primer is S: CTGCA CGTGGATGCA GAACCC,

[0079] SEQ ID NO 24, As: gaaa tcttcg agtgtga agaccattct agaGTT TAGC TGTG TTAAGGG TCA AG, SEQ ID NO 25, EGFP-F2 is a fragment containing Nluc, PCR amplification primer is S: CTTG ACCCTTAAC ACAG CTAA ACtct agaat ggtctt cacac tcgaa gatttc, SEQ ID NO 26; As: CGCatgcatcg ccaga atgcgt tcgca, SEQ ID NO 27. After the two fragments are fused by fusion PCR, they are connected into the pEV71-js1-EGFP plasmid by NotI / NsiI to replace the sequence in the original plasmid, and the pEV71-js1-Nluc plasmid is obtained.

[0080] Example 2: Replication ability and infection ability of the virus produced by the infectious cDNA clone of EV71 strain js1.

[0081] The infectious clone plasmid pEV71-js1 is linearized by HindIII, and then transcribed in vitro by using a T7 in vitro transcription kit (Ambion). The RNA3g transcribed in vitro is introduced into Vero cells by electroporation. Two days after the electroporation, when the cells show cytopathic effect, the virus supernatant is collected, centrifuged at 3000g for 10 min, and then filtered through a 0.45m filter membrane to remove cell debris. The virus in the supernatant is titrated by plaque assay. The plaques formed by the virus produced by the infectious clone plasmid are compared with the plaques of the original parent virus as shown in Figure 2 , and there is no significant difference between the morphology and size of the plaques. The virus produced by the infectious clone plasmid at the same titer is used to infect Vero cells again (MOI=0.1), and the cell supernatant collected at different times after the infection is titrated by plaque assay (expressed in PFU / ml), and the growth curves of the two are shown in Figure 2 , and there is no significant difference between the growth curves of the two.

[0082] Example 3: Production and stability of recombinant viruses containing reporter genes Nluc and EGFP.

[0083] The infectious clone plasmid containing reporter genes Nluc and EGFP is transcribed into viral RNA in vitro as above, and then electroporated into Vero cells. Two days later, the virus in the cell supernatant is collected, and the virus titer is titrated by plaque assay on Vero cells. As shown in Figure 3As shown in A, the plaque morphology and size of viruses containing the reporter genes EGFP and Nluc are similar to those of viruses without the reporter gene. Vero cells were reinfected with the same titer of the viruses containing each reporter gene and the virus without the reporter gene (MOI = 0.1). Supernatants were collected at different days after infection and titrated using a plaque assay. The resulting growth curves are shown in Figure 2. Figure 3 As shown in B, the growth cycle of the virus carrying the reporter gene is delayed compared to the wild-type virus, suggesting that the fusion of the reporter gene leads to a delay in the viral replication cycle. The replication capacity of the virus produced by the infectious clone plasmid containing the reporter gene Nluc can be determined by measuring the activity of Nluc in the cell using the Nluc substrate (Promega). The plasmid containing the VP1 E145G and 3C C147A mutations was transcribed into viral RNA in vitro. The viral RNA was introduced into Vero cells by electroporation, and the intracellular Nluc activity was measured at different times, as shown in Figure 2. Figure 5 As shown, after transfection with viral RNA containing a 3C protease inactivation mutation (C147A), the activity of Nluc in the cells no longer increased after 8 hours, and its activity only reflects the initiation of translation of viral RNA. However, after transfection with wild-type live VP145G viral RNA, Nluc gradually increased over time, indicating a normal viral replication signal. To demonstrate the stability of the reporter gene of the virus containing the EGFP reporter gene, we used the supernatant of cells infected with EV71-EGFP virus to dilute it 1:10 and reinfect new Vero cells. Two days after infection, the cells were observed under a fluorescence microscope and the supernatant was collected. New Vero cells (C+1) were reinfected again with a 1:10 dilution. Two days after infection, the cells were observed under a fluorescence microscope and the supernatant was collected for reinfection as above. The infection was sequentially passaged and the expression of EGFP in the infected cells was observed. After at least 6 generations of continuous passages, the EGFP gene remained stable.

[0084] Example 4: Infectious cDNA of EV71 strain js1 was cloned to produce virus and infect mice to establish an animal infection model.

[0085] like Figure 6 As shown in A, the virus produced by the infectious cDNA clone was used to infect 3-day-old fetal mice of different strains (1.4×104 pfu / mouse). The mice were observed 5 days after infection. Compared with uninfected mice, the mice infected with the virus all showed quadriplegia. The survival curves of the different strains of mice after infection are shown in Figure 6As shown in Table B, within 10 days, all the mice reached 100% mortality. The virus produced by the infectious clone carrying the VP1 E145G mutation did not cause the death of 3-day-old ICR mice, unlike wild-type mice, indicating that the E145 site is a decisive site for virus infection in mice. It also explains why the passage virus has a lower infection and mortality rate as the number of passages increases. SEQUENCE LISTING <110> Fudan University <120> Infectious cDNA clone based on EV71 strain and its application <130> 20190601 <160> 27 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9446 <212> DNA <213> Artificial <400> 1 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct 60 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata 120 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa 180 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa 240 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg 300 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt 360 ttcccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat 420 tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 480 ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 540 ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 600 ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 660 gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 720 gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 780 gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaaggg gtctgacgct 840 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 900 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 960 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 1020 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 1080 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 1140 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 1200 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 1260 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 1320 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 1380 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 1440 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 1500 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 1560 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 1620 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 1680 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 1740 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 1800 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 1860 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 1920 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt gtcgacgcgg 1980 ccgctaatac gactcactat aggttaaaac agcctgtggg ttgcacccac tcacagggcc 2040 tactgggcgc aagcactctg gtacctcggt acctttgtgc gcctgtttta cacccccccc 2100 ccaatgaaac ttagaagcaa taaaccacga tcaatagcag gcataacgct ccagttatgt 2160 cttgatcaag cacttctgtt tccccggact gagtatcaat agactgctcg cgcggttgaa 2220 ggagaaaacg ttcgttatcc ggctaactac ttcggaaaac ctagtaacac catgaaagtt 2280 gcggagagct tcgttcagca ctcccccagt gtagatcagg tcgatgagtc accgcgttcc 2340 ccacgggcga ccgtggcggt ggctgcgttg gcggcctgcc catggggtaa cccatggggc 2400 gctctaatac ggacatggtg tgaagagtct actgagctag ttggtagtcc tccggcccct 2460 gaatgcggct aatcccaact gcggagcaca cgcccacaag ccagcgggta gtgtgtcgta 2520 acgggtaact ctgcagcgga accgactact ttgggtgtcc gtgtttcctt ttatctttat 2580 attggctgct tatggtgaca attaaagaat tgttaccata tagctattgg attagccatc 2640 cggtgtgcaa cagagcaatt atttacctat ttattggttt tgtaccatta acctcgaatt 2700 ctgtgaccac ccttaattat atcttgaccc ttaacacagc taaacatggg ttcgcaagtg 2760 tctacacagc gctccggttc ttacgaaaac tcaaactcag ccactgaggg ttctaccata 2820 aactacacca ccattaatta ctacaaagac tcctatgctg ccacagcagg caaacagagt 2880 ctcaagcagg atccagacaa gtttgcaaat cctgttaaag acatattcac cgaaatggca 2940 gcgccactga agtccccatc cgctgaggca tgtggataca gtgatcgagt ggcgcaatta 3000 actattggca actccaccat cacgacgcaa gaagcggcta acatcatagt cggctatggt 3060 gagtggcctt cctactgctc agattctgac gctacagcag tggataaacc aacgcgcccg 3120 gatgtttcag tgaacaggtt ttacacattg gacactaaat tgtgggagaa atcgtccaag 3180 ggatggtact ggaagttccc ggatgtgtta actgaaactg gggtttttgg gcaaaatgca 3240 caattccact acctctaccg atcagggttc tgcatccacg tgcagtgcaa tgccagtaaa 3300 ttccaccaag gagcactcct agtcgctgtc ctaccagagt atgtcattgg gacagtggca 3360 ggcggtacag ggacggaaga cacccacccc ccctacaagc agacccaacc cggcgccgat 3420 ggtttcgagt tgcaacaccc gtacgtgctt gatgctggca tcccaatatc acagttaaca 3480 gtgtgcccac accagtggat taatttgagg accaacaatt gtgctacaat aatagtgcca 3540 tacattaacg cactgccttt tgattctgcc ttgaaccatt gcaactttgg cctgttagtt 3600 gtgcctatta gcccactaga ctacgaccaa ggagcaacgc cagtaatccc tataactatc 3660 acattggccc caatgtgctc tgaattcgca ggtcttaggc aggcagtcac gcaagggttc 3720 cccaccgagc taaaacctgg cacaaatcaa tttttaacca ccgatgatgg cgtctcagca 3780 cctattctac caaacttcca ccccaccccg tgtatccaca tacctggtga agttaggaac 3840 ttgctagagt tatgccaggt ggagaccatt ctggaggtta acaatgtgcc cacgaatgcc 3900 actagcttaa tggagagact gcgcttcccg gtctcagcac aagcagggaa aggtgaactg 3960 tgtgcggtgt ttagagccga tcctgggcga aatggaccat ggcaatccac cttactgggc 4020 CAGTTGTGCG GGTACTACAC CCAATGGTCA GGCTCATTGG AAGTCACCTT CATGTTTACT 4080 GGATCCTTCA TGGCTACCGG CAAGATGCTC ATAGCCTATA CACCGCCAGG GGTCTCTTG 4140 CCCAAGGACC GGGCGACCGC CATGTGGGCA CGCACGTCAT CTGGGATTTT GGGCTGCAA 4200 TCGTCTGTTA CCCTTGTAAT ACCATGGATC AGTAACACTC ATTATAGAGC ACATGCCC GA 4260 GATGGAGTGT TTGACTATTA CACTACAGGG TTAGTCAGTA TATGgtACCA GACAAATTA C 4320 GTGgtTCCAA TCGGTGCGCC CAACACAGCC TATATAATAG CACTAGCGGC AGCCCAAAG 4380 AACTTCACTA TGAAATTGTG CAAGGATGCT AGTGATATCC TGCAGACGGG CACCATCCAG 4440 GGAGATAGGG TGGCAGATGT AATTGAAAGT TCCATAGGAG ATAGCgtGA GCAGAGCCCT C 4500 ACTCACGCTC TACCAGCACC CACAGGCCAA AACACACAGT GAGCAGTCAT CGACTGGAT 4560 ACAGGCAAGG TTCCAGCACC CAAGCTGCTG AAATTGGGGC ATCATCAAAT GCTAGTGAC 4620 GAGAGCATGA TTGAAACACG TTGTGTTCTT AACTCGCATA GTACAGCTGA GACCACCTCT 4680 GATAGTTTCT TCAGTAGGGC AGGATTAGTT GGAGAGATAG ATCTCCCTCT TGAGGGCACA 4740 actaacccaa atggttatgc caactgggac atagatataa caggttacgc gcaaatgcgt 4800 agaaaggtag agctattcac ctacatgcgt tttgatgcag agttcacttt tgttgcgtgc 4860 acacccaccg gggaggttgt cccacaattg ctccaatata tgtttgtgcc acctggagcc 4920 cctaagccag attctaggga atcccttgca tggcaaaccg ccaccaaccc ctcagttttt 4980 gtcaagctgt cagaccctcc ggcgcaggtt tcagtgccat tcatgtcacc tgcgagtgct 5040 tatcaatggt tttgacgg atatcccaca ttcggagaac acaaacagga gaaagacctt 5100 gaatacgggg catgtcctaa taacatgatg ggtacattct cagtgcggac tgtggggacc 5160 tccaagtcca agtacccttt agtggttagg atttacatga gaatgaagca cgtcagggcg 5220 tggatacctc gccgatgcg caaccagaac tacctgttca aagccaaccc aaattatgct 5280 ggcaactcta ttaagccaac tggtgccagt cgcacagcga tcaccactct tgggaaattt 5340 ggacaacagt ctggggctat ttgtgggc aactttagag tggtcaaccg acatcttgcc 5400 acccataatg attgggcaaa tcttgtttgg gaagacagct ctcgcgactt gctcgtgtca 5460 tccaccactg cccaaggttg tgacacgatt gcccgttgcg attgccagac aggggtgtac 5520 tactgtaact cgatgagaaa acactaccca gtcagttttt caaaacccag cctgatctat 5580 gtagaggcta gcgagtatta cccagccagg taccaatcac atctcatgct cgcacagggt 5640 cactcggaac ctggtgattg cggtggtatc cttaggtgcc aacatggcgt catcggcata 5700 gtgtctactg gtggcaatgg gctcgttggc tttgcagacg tcagagacct cttgtggtta 5760 gatgaagaag ctatggaaca gggcgtgtcc gactacatta agggtctcgg agatgctttt 5820 ggaacaggct tcactgacgc agtctcaagg gaggttgaag ctctcaagaa ctatcttata 5880 gggtctgaag gagcagttga gaaaattttg aaaaatctta ttaaactaat ctctgcactg 5940 gtgattgtga tcagaagtga ttacgacatg gttaccctca ctgcaacctt agcgctgata 6000 ggttgtcatg gcagtccttg ggcttggatt aaagccaaaa cagcctccat cttaggtatc 6060 cctatcgccc aaaagcagag cgcttcctgg ctcaagaagt tcaatgacat ggccaacgcc 6120 gctaaggggt tagagtgggt ttccaacaag atcagcaaat ttattgattg gcttaaggag 6180 AAAATAGTAC CAGCAGCCAG GGAGAAGGTT GAATTCCTAA ATAAC TTGAA ACAGCTGCCA 6240 CTGCTAGAGA ATCAGATCTC GAACTTGGAA CAATCTGCTG CTTCAAGAAG GACCTTGA A 6300 GTGTATGTTC GGAATGTGTC GTACCTAGCT CACTTCTGTC GCAAGTTTCA ACCGCTATA C 6360 GCCACGGAAG CTAAGAAGGT CTATGCCCTG GAGAAGAGAA TGAATAACTA TATGCAGTT C 6420 AAGAGCAAAC ACCGATTGA CCCTGTATGT CTCATTATTA GGGGCTCACC AGGCACCGGG 6480 AAGTCTCTAG CC ACTG GTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTG 6540 TACTCGCTCC CACCAGACCC GGATCATT TTGACGGTTACA AGCAACAGGT G GTTACAGTG 6600 ATGGATGATT TGTTCAAAAA CCCGATGGTA AGGATATGTC CTTATTCTGT CAAATGGTA 6660 TCCACCGTAG ATTTTCATTC CACCAATGGC TTCTCTCGAG GAGAAGGGAG TTTCCTTCAC C 6720 TCTAAGTTTG T CATCGCATC CACTAATGCC AGTAATATCA TAGTACCAAC AGTGTCTGAT 6780 TCTGACGCTA TTCGCCGCAG GTTCTACATG GACTGTGACA TTGAAGTGAC AGACTC GTAC 6840 AAAACAGATC TAGGTA GACT GGATGCAGGG CGAGCCGCTA AACTGTGTTCT GAAAATAAC 6900 actgcaaatt tcaaacgttg cagcccatta gtgtgtggga aagccatcca acttagagat 6960 agaaagtcta aagtcagata cagtgtggat acggtggttt cagaacttat tagggaatac 7020 agcaataggt ccgccattgg taacacaatc gaggctcttt tccaaggtcc acccaagttc 7080 aggccaatta ggattagcct tgaagaaaaa ccagccccag acgctattag cgatctcctt 7140 gctagtgtag atagtgaaga agtgcgccag tactgcaggg atcaaggctg gattattcct 7200 gaagctccca ccaatgtgga gcggcacctt atagagcgg tgctcgtcat gcaatccatc 7260 accacagtag tggcggttgt ttcgttggtg tacgtcatct acaagctctt tgcagggttt 7320 cagggtgcat attctggtgc tcctaagcaa gtgcttaaga aacctgctct tcgcacagca 7380 acagtgcagg gtccgagcct tgactttgct ctctccctac tgagaaggaa catcaggcag 7440 gtccaaacag accaagggca tttcaccatg ttgggtgtta gggatcgctt agcagtcctc 7500 ccacgccact cacaacctgg caaaaccatt tggattgagc acaaactcgt gaacgtcctt 7560 gatgcagttg aactggtgga tgagcaagga gtcaacctgg aattaaccct catcactctt 7620 GACACCAACG AGAAGTTTAG GGATATCACC AAATTCATCC CAGAAAATAT CAGC ACTGCT 7680 AGCGATGCCA CCCTAGTGAT CAACACGGAG CACATGCCGT CAATGTTTGT CCCG GTGGGT 7740 GACGTTGTGC AGTATGGCTT TTGAATCTC AGTGGCAAGC CTACCCATCG CACCATGATG 7800 TACAATTTTC CTACTAAAGC AGGACAGTGT GGAGGAAGTG GTGACATCTG TTGGGAAGGT 7860 GTCG GTATTCACTTTG GTGGCAATGGCAGACAAGGTTTTT GC GC AGGCCT CAAAAGGAGT 7920 TACTTTGCTA GTGAACAAGG AGATCCAGTG GGTAAAGCCA ATAAGAAACT GGAAGA 7980 CTCAACATCA ATGGACCAAC CC GCACCAAGT TAGAACCTAGT GTATTCCATG ACATCTTC 8040 GAGGGAAATA AGGAACCAGC TGTCCTGCAC AGTAAAGACC CCCGACTTGA GGTA GTATT 8100 GAACAGGCCCT GTTCTCTAAGT ATGTGGGAAAC ACACACTAC ATGAGCCTGA CGAGTACAT 8160 AAAGAGGCAG CTCTACATTA TGC AACCAATTA AAGCAACTAG AAATCAATAC CTCTCAA 8220 ATGAGCATGG AGGAGGCCTG CTATGGTACT GAGAATCTTG AGGCTATTC ATCTTCACAC 8280 AGTGCAGGTT ACCCCTATAG TGCCCTAGGG ATAAAGAAAA GAGACATCTT AGACCCTACC 8340 accagggacg tgagtagaat gaagttctac atggacaagt atggtcttga tcttccctac 8400 tccacttatg tcaaggacga gctacgctcg attgataaaa tcaagaaagg gaagtcccgc 8460 ctgatcgagg ccagtagtct aaatgattca gtgtacctca gaatggcttt cgggcatttg 8520 tatgaggctt tccacgcaaa tcctgggacg ataactggat cggccgtggg gtgtaaccct 8580 gacacattct ggagcaagct gccaattttg ctccctggtt cactctttgc ctttgactac 8640 tcaggctatg atgccagcct tagccctgtc tggttcagag cattagaatt ggttcttagg 8700 gagatagggt atagtgaaga ggcaatctca ctcattgagg gaatcaacca cacacatcat 8760 gtgtatcgta ataagaccta ttgcgtgctt ggtgggatgc cctcaggctg ttcaggaaca 8820 tccatcttca actcaatgat caacaacatt attatcagag cactgctcat aaaaacattt 8880 aagggcattg atttggatga actcaacatg gtcgcttatg gagacgatgt gctcgctagc 8940 tatcccttcc caattgattg cttggaacta gcaaagactg gtaaggagta tggtctgacc 9000 atgacccctg ctgataaatc tccttgcttt aatgaggtca attggggtaa tgcgaccttc 9060 ctcaaaaggg gctttttgcc cgatgaacag tttccatttt tgattcaccc tactatgcca 9120 atgagggaga tccatgagtc cattcgatgg accaaggacg cacggaacac tcaagatcat 9180 gtgcggtcct tgtgcctcct agcatggcat aatggtaagc aagaatacga gaagtttgtg 9240 agcacaatta ggtctgtccc agtagggaga gcgttggcta ttccaaatta tgaaaatctt 9300 agacgaaatt ggctcgagtt attttagagg ttatacacac ctcaacccca ccagaaatct 9360 ggtcgtgaat gtgactggtg ggggtaaatt tgttataacc agaatagcaa aaaaaaaaaa 9420 aaaaaaaaaa aaaaaaaaaa gcttat 9446 <210> 2 <211> 7405 <212> DNA <213> Artificial <400> 2 ttaaaacagc ctgtgggttg cacccactca cagggcctac tgggcgcaag cactctggta 60 cctcggtacc tttgtgcgcc tgttttacac ccccccccca atgaaactta gaagcaataa 120 accacgatca atagcaggca taacgctcca gttatgtctt gatcaagcac ttctgtttcc 180 ccggactgag tatcaataga ctgctcgcgc ggttgaagga gaaaacgttc gttatccggc 240 taactacttc ggaaaaccta gtaacaccat gaaagttgcg gagagcttcg ttcagcactc 300 ccccagtgta gatcaggtcg atgagtcacc gcgttcccca cgggcgaccg tggcggtggc 360 tgcgttggcg gcctgcccat ggggtaaccc atggggcgct ctaatacgga catggtgtga 420 agagtctact gagctagttg gtagtcctcc ggcccctgaa tgcggctaat cccaactgcg 480 gagcacacgc ccacaagcca gcgggtagtg tgtcgtaacg ggtaactctg cagcggaacc 540 gactactttg ggtgtccgtg tttcctttta tctttatatt ggctgcttat ggtgacaatt 600 aaagaattgt taccatatag ctattggatt agccatccgg tgtgcaacag agcaattatt 660 tacctattta ttggttttgt accattaacc tcgaattctg tgaccaccct taattatatc 720 ttgaccctta acacagctaa acatgggttc gcaagtgtct acacagcgct ccggttctta 780 cgaaaactca aactcagcca ctgagggttc taccataaac tacaccacca ttaattacta 840 caaagactcc tatgctgcca cagcaggcaa acagagtctc aagcaggatc cagacaagtt 900 tgcaaatcct gttaaagaca tattcaccga aatggcagcg ccactgaagt ccccatccgc 960 TGAGGAGAAG GAGAAGAAGA AGAAGAAGAA GAAGAAGGAA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAAGAAGAAG 60 GACGCAAGAA GCAGCTAACA TCACTGTGGC TATGGTGAGT GGCCTTCCTA CTGCTCAGA 1080 TTCTGACGCT ACAGCAGTGG ATAAACCAAC GCACCCGGAT GTTTCAGTGA ACAGGTCTTA 1140 CACATTGGAC ACTAAATGT GGGAGAAATC GTCCAAGGAT GGTACTGGAA GTTCCCGGA 1200 TGTGTAACT GAAACTGGGG TTTTTGGGCA AAATGCACAA TTCCACTACC TCTACC GATC 1260 AGGGTTCTGC ATCCACGTGC AGTGCAATGC CAGTA AATT CCACCAAGGA GC ACTCCTAGT 1320 CGCTGTCCTA CCAGAGTATG TCATTGGGAC AGTGGCAGGC GGTACAGGAC GGAAGACAC 1380 CCACCCCCCC TACAAGCAGA CCCAACCCGG CGCCGATGGT TTCGAGTTGC AACACCCGTA 1440 Cgtgcttgat gctggcatcc caatatcaca gttaacagtg tgcccacacc agtggattaa 1500 TTTGAGGACC AACAATTGTG CTACAATAAT AGTGCCATA CATTAAAGCA CTGCCTTTGA 1560 TTCTGCCTTG AACCATTGCA ACTTTGGCCT GTTAGTTGTG CCTATTAGCC CACTAGACTA 1620 CGACCAAGGA GC AACGCCAGT AATCCCTATA ACTATCACA TTGGCCCCAA TGTGCTCTGA 1680 attcgcaggt cttaggcagg cagtcacgca agggttcccc accgagctaa aacctggcac 1740 aaatcaattt ttaaccaccg atgatggcgt ctcagcacct attctaccaa acttccacccc 1800 caccccgtgt atccacatac ctggtgaagt taggaacttg ctagagttat gccaggtgga 1860 gaccattctg gaggttaaca atgtgcccac gaatgccact agcttaatgg agagactgcg 1920 cttcccggtc tcagcacaag cagggaaagg tgaactgtgt gcggtgttta gagccgatcc 1980 tgggcgaaat ggaccatggc aatccacctt actgggccag ttgtgcgggt actacaccca 2040 atggtcaggg tcattggaag tcaccttcat gttactgga tccttcatgg ctaccggcaa 2100 gatgctcata gcctatacac cgccaggggg tcctctgccc aaggaccggg cgaccgccat 2160 gttgggcacg cacgtcatct gggattttgg gctgcaatcg tctgttaccc ttgtaatacc 2220 atggatcagt aacactcatt atagagcaca tgcccgagat ggagtgtttg actattacac 2280 tacagggtta gtcagtatat ggtaccagac aaattacgtg gttccaatcg gtgcgcccaa 2340 cacagcctat ataatagcac tagcggcagc ccaaaagaac ttcactatga aattgtgcaa 2400 ggatgctagt gatatcctgc agacgggcac catccaggga gatagggtgg cagatgtaat 2460 tgaaagttcc ataggagata gcgtgagcag agccctcact cacgctctac cagcacccac 2520 aggccaaaac acacaggtga gcagtcatcg actggataca ggcaaggttc cagcactcca 2580 agctgctgaa attggggcat catcaaatgc tagtgacgag agcatgattg aaacacgttg 2640 tgttcttaac tcgcatagta cagctgagac cactcttgat agtttcttca gtagggcagg 2700 attagttgga gagatagatc tccctcttga gggcacaact aacccaaatg gttatgccaa 2760 ctgggacata gatataacag gttacgcgca aatgcgtaga aaggtagagc tattcaccta 2820 catgcgtttt gatgcagagt tcacttttgt tgcgtgcaca cccaccgggg aggttgtccc 2880 acaattgctc caatatatgt ttgtgccacc tggagcccct aagccagatt ctagggaatc 2940 ccttgcatgg caaaccgcca ccaacccctc agtttttgtc aagctgtcag accctccggc 3000 gcaggtttca gtgccattca tgtcacctgc gagtgcttat caatggtttt atgacggata 3060 tcccacattc ggagaacaca aacaggagaa agaccttgaa tacggggcat gtcctaataa 3120 catgatgggt acattctcag tgcggactgt ggggacctcc aagtccaagt accctttagt 3180 ggttaggatt tacatgagaa tgaagcacgt cagggcgtgg atacctcgcc cgatgcgcaa 3240 ccagaactac ctgttcaaag ccaacccaaa ttatgctggc aactctatta agccaactgg 3300 tgccagtcgc acagcgatca ccactcttgg gaaatttgga caacagtctg gggctattta 3360 tgtgggcaac tttagagtgg tcaaccgaca tcttgccacc cataatgatt gggcaaatct 3420 tgtttgggaa gacagctctc gcgacttgct cgtgtcatcc accactgccc aaggttgtga 3480 cacgattgcc cgttgcgatt gccagacagg ggtgtactac tgtaactcga tgagaaaaca 3540 ctacccagtc agtttttcaa aacccagcct gatctatgta gaggctagcg agtattaccc 3600 agccaggtac caatcacatc tcatgctcgc acagggtcac tcggaacctg gtgattgcgg 3660 tggtatcctt aggtgccaac atggcgtcat cggcatagtg tctactggtg gcaatgggct 3720 cgttggcttt gcagacgtca gagacctctt gtggttagat gaagaagcta tggaacaggg 3780 cgtgtccgac tacattaagg gtctcggaga tgcttttgga acaggcttca ctgacgcagt 3840 ctcaagggag gttgaagctc tcaagaacta tcttataggg tctgaaggag cagttgagaa 3900 aattttgaaa aatcttatta aactaatctc tgcactggtg attgtgatca gaagtgatta 3960 cgacatggtt accctcactg caaccttagc gctgataggt tgtcatggca gtccttgggc 4020 ttggattaaa gccaaaacag cctccatctt aggtatccct atcgcccaaa agcagagcgc 4080 ttcctggctc aagaagttca atgacatggc caacgccgct aaggggttag agtgggtttc 4140 caacaagatc agcaaattta ttgattggct taaggagaaa atagtaccag cagccaggga 4200 gaaggttgaa ttcctaaata acttgaaaca gctgccactg ctagagaatc agatctcgaa 4260 cttggaacaa tctgctgctt cacaagagga ccttgaagtc atgtttggga atgtgtcgta 4320 cctagctcac ttctgtcgca agtttcaacc gctatacgcc acggaagcta aaagagtcta 4380 tgccctggag aagagaatga ataactatat gcagttcaag agcaaacacc gaattgaacc 4440 tgtatgtctc attattaggg gctcaccagg caccgggaag tctctagcca ctggtattat 4500 tgctcgagca atcgctgata agtaccactc cagcgtgtac tcgctcccac cagacccgga 4560 tcattttgac ggttacaagc aacaggtggt tacagtgatg gatgatttgt gtcaaaaccc 4620 cgatggtaag gatatgtcct tattctgtca aatggtatcc accgtagatt tcattccacc 4680 aatggcttct ctcgaggaga agggagtttc cttcacctct aagtttgtca tcgcatccac 4740 taatgccagt aatatcatag taccaacagt gtctgattct gacgctattc gccgcaggtt 4800 ctacatggac tgtgacattg aagtgacaga ctcgtacaaa acagatctag gtagactgga 4860 tgcagggcga gccgctaaac tgtgttctga aaataacact gcaaatttca aacgttgcag 4920 cccattagtg tgtgggaaag ccatccaact tagagataga aagtctaaag tcagatacag 4980 tgtggatacg gtggtttcag aacttattag ggaatacagc aataggtccg ccattggtaa 5040 cacaatcgag gctcttttcc aaggtccacc caagttcagg ccaattagga ttagccttga 5100 agaaaaacca gccccagacg ctattagcga tctccttgct agtgtagata gtgaagaagt 5160 gcgccagtac tgcagggatc aaggctggat tattcctgaa gctcccacca atgtggagcg 5220 gcaccttaat agagcggtgc tcgtcatgca atccatcacc acagtagtgg cggttgtttc 5280 gttggtgtac gtcatctaca agctctttgc agggtttcag ggtgcatatt ctggtgctcc 5340 5400 ctttgctctc tccctactga gaaggaacat caggcaggtc caaacagacc aagggcattt 5460 caccatgttg ggtgttaggg atcgcttagc agtcctccca cgccactcac aacctggcaa 5520 aaccatttgg attgagcaca aactcgtgaa cgtccttgat gcagttgaac tggtggatga 5580 5640 tatcaccaaa ttcatcccag aaaatatcag cactgctagc gatgccaccc tagtgatcaa 5700 cacggagcac atgccgtcaa tgtttgtccc ggtgggtgac gttgtgcagt atggcttttt 5760 gaatctcagt ggcaagccta cccatcgcac catgatgtac aattttccta ctaaagcagg 5820 acagtgtgga ggagtggtga catctgttgg gaaggttgtc ggtattcaca ttggtggcaa 5880 tggcagacaa ggtttttgcg caggcctcaa aaggattac tttgctagtg aacaaggaga 5940 gatccagtgg gttaagccca ataaagaaac tggagaactc aacatcaatg gaccaacccg 6000 caccaagtta gaacctagtg tattccatga catcttcag ggaaataagg aaccagctgt 6060 cttgcacagt aaagaccccc gacttgaggt agattttgaa caggccctgt tctctaagta 6120 tgtgggaaac acactacatg agcctgacga gtacatcaaa gaggcagctc tacattatgc 6180 aaaccaatta aagcaactag aaatcaatac ctctcaaatg agcatggagg aggcctgcta 6240 tggtactgag aatcttgagg ctattgatct tcacactagt gcaggttacc cctatagtgc 6300 cctagggata aagaaaagag acatcttaga ccctaccacc agggacgtga gtagaatgaa 6360 gttctacatg gacaagtatg gtcttgatct tccctactcc acttatgtca aggacgagct 6420 acgctcgatt gataaaatca agaaagggaa gtcccgcctg atcgaggcca gtagtctaaa 6480 tgattcagtg tacctcagaa tggctttcgg gcatttgtat gaggctttcc acgcaaatcc 6540 tgggacgata actggatcgg ccgtggggtg taaccctgac acattctgga gcaagctgcc 6600 aattttgctc cctggttcac tctttgcctt tgactactca ggctatgatg ccagccttag 6660 ccctgtctgg ttcagagcat tagaattggt tcttagggag atagggtata gtgaagaggc 6720 aatctcactc attgagggaa tcaaccacac acatcatgtg tatcgtaata agacctattg 6780 cgtgcttggt gggatgccct caggctgttc aggaacatcc atcttcaact caatgatcaa 6840 caacattatt atcagagcac tgctcataaa aacatttaag ggcattgatt tggatgaact 6900 caacatggtc gcttatggag acgatgtgct cgctagctat cccttcccaa ttgattgctt 6960 ggaactagca aagactggta aggagtatgg tctgaccatg acccctgctg ataaatctcc 7020 ttgctttaat gaggtcaatt ggggtaatgc gaccttcctc aaaaggggct ttttgcccga 7080 tgaacagttt ccatttttga ttcaccctac tatgccaatg agggagatcc atgagtccat 7140 tcgatggacc aaggacgcac ggaacactca agatcatgtg cggtccttgt gcctcctagc 7200 atggcataat ggtaagcaag aatacgagaa gtttgtgagc acaattaggt ctgtcccagt 7260 agggagagcg ttggctattc caaattatga aaatcttaga cgaaattggc tcgagttatt 7320 ttagaggtta tacacacctc aaccccacca gaaatctggt cgtgaatgtg actggtgggg 7380 gtaaatttgt tataaccaga atagc 7405 <210> 3 <211> 1987 <212> DNA <213> Artificial <400> 3 agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt 60 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg 120 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg 180 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg 240 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc 300 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg 360 cgtttcccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt 420 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg 480 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct 540 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag 600 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa 660 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag 720 ttggtagctc agaaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag 780 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa ggggtctgac 840 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 900 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 960 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1020 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1080 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 1140 gatttatcag aataaacca gccagccgga agggccgagc gcaagagtgg tcctgcaact 1200 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtag tagttcgcca 1260 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg 1320 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatcccc 1380 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 1440 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 1500 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 1560 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc 1620 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 1680 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 1740 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 1800 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 1860 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 1920 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtgtcgacg 1980 cggccgc 1987 <210> 4 <211> 2193 <212> PRT <213> Artificial <400> 4 Met Gly Ser Gin Val Ser Thr Gin Arg Ser Gly Ser Tyr Glu Asn Ser 1 5 10 15 Asn Ser Ala Thr Glu Gly Ser Thr Ile Asn Tyr Thr Thr Ile Asn Tyr 20 25 30 Tyr Lys Asp Ser Tyr Ala Ala Thr Ala Gly Lys Gin Ser Leu Lys Gin 35 40 45 Asp Pro Asp Lys Phe Ala Asn Pro Val Lys Asp Ile Phe Thr Glu Met 50 55 60 Ala Ala Pro Leu Lys Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp 65 70 75 80 Arg Val Ala Gin Leu Thr Ile Gly Asn Ser Thr Ile Thr Thr Gin Glu 85 90 95 Ala Ala Asn Ile Ile Val Gly Tyr Gly Glu Trp Pro Ser Tyr Cys Ser 100 105 110 Asp Ser Asp Ala Thr Ala Val Asp Lys Pro Thr Arg Pro Asp Val Ser 115 120 125 Val Asn Arg Phe Tyr Thr Leu Asp Thr Lys Leu Trp Glu Lys Ser Ser 130 135 140 Lys Gly Trp Tyr Trp Lys Phe Pro Asp Val Leu Thr Glu Thr Gly Val 145 150 155 160 Phe Gly Gin Asn Ala Gin Phe His Tyr Leu Tyr Arg Ser Gly Phe Cys 165 170 175 Ile His Val Gin Cys Asn Ala Ser Lys Phe His Gin Gly Ala Leu Leu 180 185 190 Val Ala Val Leu Pro Gin Tyr Val He Gly Thr Val Ala Gly Gly Thr 195 200 205 Gly Thr Gin Asp Thr His Pro Pro Tyr Gin Gin Thr Gin Pro Gly Ala 210 215 220 Asp Gly Phe Gin Leu Gin His Pro Tyr Val Leu Asp Ala Gly He Pro 225 230 235 240 Ile Ser Gin Leu Thr Val Cys Pro His Gin Trp He Asn Leu Arg Thr 245 250 255 Asn Asn Cys Ala Thr He He Val Pro Tyr He Asn Ala Leu Pro Phe 260 265 270 Asp Ser Ala Leu Asn His Cys Asn Phe Gly Leu Leu Val Val Pro He 275 280 285 Ser Pro Leu Asp Tyr Asp Gin Gly Ala Thr Pro Val He Pro He Thr 290 295 300 Ile Thr Leu Ala Pro Met Cys Ser Gin Phe Ala Gly Leu Arg Gin Ala 305 310 315 320 Val Thr Gin Gly Phe Pro Thr Glu Leu Lys Pro Gly Thr Asn Gin Phe 325 330 335 Leu Thr Thr Asp Asp Gly Val Ser Ala Pro Ile Leu Pro Asn Phe His 340 345 350 Pro Thr Pro Cys Ile His Ile Pro Gly Glu Val Arg Asn Leu Leu Glu 355 360 365 Leu Cys Gin Val Glu Thr Ile Leu Glu Val Asn Asn Val Pro Thr Asn 370 375 380 Ala Thr Ser Leu Met Glu Arg Leu Arg Phe Pro Val Ser Ala Gin Ala 385 390 395 400 Gly Lys Gly Glu Leu Cys Ala Val Phe Arg Ala Asp Pro Gly Arg Asn 405 410 415 Gly Pro Trp Gin Ser Thr Leu Leu Gly Gin Leu Cys Gly Tyr Tyr Thr 420 425 430 Gln Trp Ser Gly Ser Leu Glu Val Thr Phe Met Phe Thr Gly Ser Phe 435 440 445 Met Ala Thr Gly Lys Met Leu Ile Ala Tyr Thr Pro Pro Gly Gly Pro 450 455 460 Leu Pro Lys Asp Arg Ala Thr Ala Met Leu Gly Thr His Val Ile Trp 465 470 475 480 Asp Phe Gly Leu Gln Ser Ser Val Thr Leu Val Ile Pro Trp Ile Ser 485 490 495 Asn Thr His Tyr Arg Ala His Ala Arg Asp Gly Val Phe Asp Tyr Tyr 500 505 510 Thr Thr Gly Leu Val Ser Ile Trp Tyr Gln Thr Asn Tyr Val Val Pro 515 520 525 Ile Gly Ala Pro Asn Thr Ala Tyr Ile Ile Ala Leu Ala Ala Ala Gln 530 535 540 Lys Asn Phe Thr Met Lys Leu Cys Lys Asp Ala Ser Asp Ile Leu Gln 545 550 555 560 Thr Gly Thr Ile Gln Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser 565 570 575 Ile Gly Asp Ser Val Ser Arg Ala Leu Thr His Ala Leu Pro Ala Pro 580 585 590 Thr Gly Gln Asn Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys 595 600 605 Val Pro Ala Leu Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser 610 615 620 Asp Glu Ser Met Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr 625 630 635 640 Ala Glu Thr Thr Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val Gly 645 650 655 Glu Ile Asp Leu Pro Leu Glu Gly Thr Thr Asn Pro Asn Gly Tyr Ala 660 665 670 Asn Trp Asp Ile Asp Ile Thr Gly Tyr Ala Gln Met Arg Arg Lys Val 675 680 685 Glu Leu Phe Thr Tyr Met Arg Phe Asp Ala Glu Phe Thr Phe Val Ala 690 695 700 Cys Thr Pro Thr Gly Glu Val Val Pro Gln Leu Leu Gln Tyr Met Phe 705 710 715 720 Val Pro Pro Gly Ala Pro Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp 725 730 735 Gln Thr Ala Thr Asn Pro Ser Val Phe Val Lys Leu Ser Asp Pro Pro 740 745 750 Ala Gln Val Ser Val Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp 755 760 765 Phe Tyr Asp Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp 770 775 780 Leu Glu Tyr Gly Ala Cys Pro Asn Asn Met Met Gly Thr Phe Ser Val 785 790 795 800 Arg Thr Val Gly Thr Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile 805 810 815 Tyr Met Arg Met Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg 820 825 830 Asn Gln Asn Tyr Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser 835 840 845 Ile Lys Pro Thr Gly Ala Ser Arg Thr Ala Ile Thr Thr Leu Gly Lys 850 855 860 Phe Gly Gln Gln Ser Gly Ala Ile Tyr Val Gly Asn Phe Arg Val Val 865 870 875 880 Asn Arg His Leu Ala Thr His Asn Asp Trp Ala Asn Leu Val Trp Glu 885 890 895 Asp Ser Ser Arg Asp Leu Leu Val Ser Ser Thr Thr Ala Gln Gly Cys 900 905 910 Asp Thr Ile Ala Arg Cys Asp Cys Gln Thr Gly Val Tyr Tyr Cys Asn 915 920 925 Ser Met Arg Lys His Tyr Pro Val Ser Phe Ser Lys Pro Ser Leu Ile 930 935 940 Tyr Val Glu Ala Ser Glu Tyr Tyr Pro Ala Arg Tyr Gin Ser His Leu 945 950 955 960 Met Leu Ala Gin Gly His Ser Glu Pro Gly Asp Cys Gly Gly Ile Leu 965 970 975 Arg Cys Gin His Gly Val Ile Gly Ile Val Ser Thr Gly Gly Asn Gly 980 985 990 Leu Val Gly Phe Ala Asp Val Arg Asp Leu Leu Trp Leu Asp Glu Glu 995 1000 1005 Ala Met Glu Gin Gly Val Ser Asp Tyr Ile Lys Gly Leu Gly Asp Ala 1010 1015 1020 Phe Gly Thr Gly Phe Thr Asp Ala Val Ser Arg Glu Val Glu Ala Leu 1025 1030 1035 1040 Lys Asn Tyr Leu Ile Gly Ser Glu Gly Ala Val Glu Lys Ile Leu Lys 1045 1050 1055 Asn Leu Ile Lys Leu Ile Ser Ala Leu Val Ile Val Ile Arg Ser Asp 1060 1065 1070 Tyr Asp Met Val Thr Leu Thr Ala Thr Leu Ala Leu Ile Gly Cys His 1075 1080 1085 Gly Ser Pro Trp Ala Trp Ile Lys Ala Lys Thr Ala Ser Ile Leu Gly 1090 1095 1100 Ile Pro Ile Ala Gln Lys Gln Ser Ala Ser Trp Leu Lys Lys Phe Asn 1105 1110 1115 1120 Asp Met Ala Asn Ala Ala Lys Gly Leu Glu Trp Val Ser Asn Lys Ile 1125 1130 1135 Ser Lys Phe Ile Asp Trp Leu Lys Glu Lys Ile Val Pro Ala Ala Arg 1140 1145 1150 Glu Lys Val Glu Phe Leu Asn Asn Leu Lys Gln Leu Pro Leu Leu Glu 1155 1160 1165 Asn Gln Ile Ser Asn Leu Glu Gln Ser Ala Ala Ser Gln Glu Asp Leu 1170 1175 1180 Glu Val Met Phe Gly Asn Val Ser Tyr Leu Ala His Phe Cys Arg Lys 1185 1190 1195 1200 Phe Gln Pro Leu Tyr Ala Thr Glu Ala Lys Arg Val Tyr Ala Leu Glu 1205 1210 1215 Lys Arg Met Asn Asn Tyr Met Gln Phe Lys Ser Lys His Arg Ile Glu 1220 1225 1230 Pro Val Cys Leu Ile Ile Arg Gly Ser Pro Gly Thr Gly Lys Ser Leu 1235 1240 1245 Ala Thr Gly lie lie Ala Arg Ala lie Ala Asp Lys Tyr His Ser Ser 1250 1255 1260 Val Tyr Ser Leu Pro Pro Asp Pro Asp His Phe Asp Gly Tyr Lys Gin 1265 1270 1275 1280 Gln Val Val Thr Val Met Asp Asp Leu Cys Gin Asn Pro Asp Gly Lys 1285 1290 1295 Asp Met Ser Leu Phe Cys Gin Met Val Ser Thr Val Asp Phe lie Pro 1300 1305 1310 Pro Met Ala Ser Leu Glu Glu Lys Gly Val Ser Phe Thr Ser Lys Phe 1315 1320 1325 Val lie Ala Ser Thr Asn Ala Ser Asn lie lie Val Pro Thr Val Ser 1330 1335 1340 Asp Ser Asp Ala lie Arg Arg Arg Phe Tyr Met Asp Cys Asp lie Glu 1345 1350 1355 1360 Val Thr Asp Ser Tyr Lys Thr Asp Leu Gly Arg Leu Asp Ala Gly Arg 1365 1370 1375 Ala Ala Lys Leu Cys Ser Glu Asn Asn Thr Ala Asn Phe Lys Arg Cys 1380 1385 1390 Ser Pro Leu Val Cys Gly Lys Ala lie Gin Leu Arg Asp Arg Lys Ser 1395 1400 1405 Lys Val Arg Tyr Ser Val Asp Thr Val Val Ser Glu Leu Ile Arg Glu 1410 1415 1420 Tyr Ser Asn Arg Ser Ala Ile Gly Asn Thr Ile Glu Ala Leu Phe Gln 1425 1430 1435 1440 Gly Pro Pro Lys Phe Arg Pro Ile Arg Ile Ser Leu Glu Glu Lys Pro 1445 1450 1455 Ala Pro Asp Ala Ile Ser Asp Leu Leu Ala Ser Val Asp Ser Glu Glu 1460 1465 1470 Val Arg Gln Tyr Cys Arg Asp Gln Gly Trp Ile Ile Pro Glu Ala Pro 1475 1480 1485 Thr Asn Val Glu Arg His Leu Asn Arg Ala Val Leu Val Met Gln Ser 1490 1495 1500 Ile Thr Thr Val Val Ala Val Val Ser Leu Val Tyr Val Ile Tyr Lys 1505 1510 1515 1520 Leu Phe Ala Gly Phe Gln Gly Ala Tyr Ser Gly Ala Pro Lys Gln Val 1525 1530 1535 Leu Lys Lys Pro Ala Leu Arg Thr Ala Thr Val Gln Gly Pro Ser Leu 1540 1545 1550 Asp Phe Ala Leu Ser Leu Leu Arg Arg Asn Ile Arg Gin Val Gin Thr 1555 1560 1565 Asp Gin Gly His Phe Thr Met Leu Gly Val Arg Asp Arg Leu Ala Val 1570 1575 1580 Leu Pro Arg His Ser Gin Pro Gly Lys Thr Ile Trp Ile Glu His Lys 1585 1590 1595 1600 Leu Val Asn Val Leu Asp Ala Val Glu Leu Val Asp Glu Gin Gly Val 1605 1610 1615 Asn Leu Glu Leu Thr Leu Ile Thr Leu Asp Thr Asn Glu Lys Phe Arg 1620 1625 1630 Asp Ile Thr Lys Phe Ile Pro Glu Asn Ile Ser Thr Ala Ser Asp Ala 1635 1640 1645 Thr Leu Val Ile Asn Thr Glu His Met Pro Ser Met Phe Val Pro Val 1650 1655 1660 Gly Asp Val Val Gin Tyr Gly Phe Leu Asn Leu Ser Gly Lys Pro Thr 1665 1670 1675 1680 His Arg Thr Met Met Tyr Asn Phe Pro Thr Lys Ala Gly Gin Cys Gly 1685 1690 1695 Gly Val Val Thr Ser Val Gly Lys Val Val Gly Ile His Ile Gly Gly 1700 1705 1710 Asn Gly Arg Gin Gly Phe Cys Ala Gly Leu Lys Arg Ser Tyr Phe Ala 1715 1720 1725 Ser Glu Gin Gly Glu lie Gin Trp Val Lys Pro Asn Lys Glu Thr Gly 1730 1735 1740 Arg Leu Asn lie Asn Gly Pro Thr Arg Thr Lys Leu Glu Pro Ser Val 1745 1750 1755 1760 Phe His Asp lie Phe Glu Gly Asn Lys Glu Pro Ala Val Leu His Ser 1765 1770 1775 Lys Asp Pro Arg Leu Glu Val Asp Phe Glu Gin Ala Leu Phe Ser Lys 1780 1785 1790 Tyr Val Gly Asn Thr Leu His Glu Pro Asp Glu Tyr lie Lys Glu Ala 1795 1800 1805 Ala Leu His Tyr Ala Asn Gin Leu Lys Gin Leu Glu lie Asn Thr Ser 1810 1815 1820 Gln Met Ser Met Glu Glu Ala Cys Tyr Gly Thr Glu Asn Leu Glu Ala 1825 1830 1835 1840 lie Asp Leu His Thr Ser Ala Gly Tyr Pro Tyr Ser Ala Leu Gly lie 1845 1850 1855 Lys Lys Arg Asp lie Leu Asp Pro Thr Thr Arg Asp Val Ser Arg Met 1860 1865 1870 Lys Phe Tyr Met Asp Lys Tyr Gly Leu Asp Leu Pro Tyr Ser Thr Tyr 1875 1880 1885 Val Lys Asp Glu Leu Arg Ser lie Asp Lys lie Lys Lys Gly Lys Ser 1890 1895 1900 Arg Leu lie Glu Ala Ser Ser Leu Asn Asp Ser Val Tyr Leu Arg Met 1905 1910 1915 1920 Ala Phe Gly His Leu Tyr Glu Ala Phe His Ala Asn Pro Gly Thr lie 1925 1930 1935 Thr Gly Ser Ala Val Gly Cys Asn Pro Asp Thr Phe Trp Ser Lys Leu 1940 1945 1950 Pro lie Leu Leu Pro Gly Ser Leu Phe Ala Phe Asp Tyr Ser Gly Tyr 1955 1960 1965 Asp Ala Ser Leu Ser Pro Val Trp Phe Arg Ala Leu Glu Leu Val Leu 1970 1975 1980 Arg Glu lie Gly Tyr Ser Glu Glu Ala lie Ser Leu lie Glu Gly lie 1985 1990 1995 2000 Asn His Thr His His Val Tyr Arg Asn Lys Thr Tyr Cys Val Leu Gly 2005 2010 2015 Gly Met Pro Ser Gly Cys Ser Gly Thr Ser Ile Phe Asn Ser Met Ile 2020 2025 2030 Asn Asn Ile Ile Ile Arg Ala Leu Leu Ile Lys Thr Phe Lys Gly Ile 2035 2040 2045 Asp Leu Asp Glu Leu Asn Met Val Ala Tyr Gly Asp Asp Val Leu Ala 2050 2055 2060 Ser Tyr Pro Phe Pro Ile Asp Cys Leu Glu Leu Ala Lys Thr Gly Lys 2065 2070 2075 2080 Glu Tyr Gly Leu Thr Met Thr Pro Ala Asp Lys Ser Pro Cys Phe Asn 2085 2090 2095 Glu Val Asn Trp Gly Asn Ala Thr Phe Leu Lys Arg Gly Phe Leu Pro 2100 2105 2110 Asp Glu Gln Phe Pro Phe Leu Ile His Pro Thr Met Pro Met Arg Glu 2115 2120 2125 Ile His Glu Ser Ile Arg Trp Thr Lys Asp Ala Arg Asn Thr Gln Asp 2130 2135 2140 His Val Arg Ser Leu Cys Leu Leu Ala Trp His Asn Gly Lys Gln Glu 2145 2150 2155 2160 Tyr Glu Lys Phe Val Ser Thr Ile Arg Ser Val Pro Val Gly Arg Ala 2165 2170 2175 Leu Ala Ile Pro Asn Tyr Glu Asn Leu Arg Arg Asn Trp Leu Glu Leu 2180 2185 2190 Phe <210> 5 <211> 9982 <212> DNA <213> Artificial <400> 5 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct 60 tcatgtggca ggaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata 120 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa 180 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacaggggaa 240 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg 300 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt 360 ttcccctggc ggctccctcg tgcgctctcc tgttcctgcc ttcggttta ccggtgtcat 420 tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 480 ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 540 ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 600 ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 660 gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 720 gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 780 gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaaggg gtctgacgct 840 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 900 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 960 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 1020 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 1080 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 1140 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 1200 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 1260 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 1320 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 1380 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 1440 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 1500 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 1560 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 1620 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 1680 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 1740 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 1800 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 1860 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 1920 AAATAGGGGTTCCGCGCACATTTCCC CGAAAAGTGCCCTGACGTGTCGACGC 1980 CCTAATACTGACTC ACTAT AGGT TAAAACGCCTGTGGGTTGCACCAC TCACAGGGCC 2040 TACTGGGCACAAGC ACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTATACCCCCCCC 2100 CCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGT 2160 CTTGATCAAGC ACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAA 2220 GGAGAAAACGTTCTGTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTT 2280 GCGGAGAGCTTCGTTCA GC ACTCCCCCAGTG TAGATCAGGTCGATGAGTC ACCGC GTTCC 2340 CCACGGGCACCGTGGCGGTGGCTGC GTTGGCGGCCTGC C ATGGGGTAACCCATGGGGC 2400 GCTCTAATACTGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCC TCCGGCCCCT 2460 GAA TGC GGCTAATCCCAACTGC GGAGC ACACGCCCACAAGCCAGCGGGTAGTGTGTCGTA 2520 ACGGGTAAC TCTGCAGCGGAACC GACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTAT 2580 ATTGGCTGCTTATGGTGACAAT TAAAGAATTGT TACC ATATAGCTATTGGAT TAGCCATC 2640 CGGTGTGCAA CAGAGCAATT ATTTACCTAT TTATTGGTTT TGTACCATTA ACCTCGAATT 2700 CTGTGACCAC CCTTAATTAT ATCTTGACCC TTAACACAGC TAAACTCTAG AATG GTCTTC 2760 ACACTCGAAG ATTCGTTGGG GACTGGCGAC AGACAGCCG GCTACAACCT GGACCAAGTC 2820 CTTGAACAGG GAGGTGTGTC CAGTTTGTTC AGAATTCGGG GTGTCCGTAA CTCCGATC 2880 CAAAGGATTG TCCTGAGCGG TGAAAATGGG CTGAAGATCG ACATCCATGT CATCATCCCG 2940 TATGAAGGTC TGAGCGGCGA CCAATGGGCC AGATCGAAAA AATTTTTAAG GTGGTGACA 3000 CCTGTGGATG ATCATCACTT TAAGGTGATC CTGC ACTATG GCACACTGGTA ATCGACGGG 3060 GTTACGCCGA ACATGATCGA CTATTTCGGA CGGCCGTATG AAGGATATCG CCTGTTCGAC 3120 GGCAAAAAGA TC ACTGTAAC AGGGACCCTG TGGAACGGCA AAAATATCGA CGAGCGC 3180 CTGATCAACC CCGACGGCTC CCTGCTGTTT CGAGTAACCA TCAACGGAGT GACC GGCTGG 3240 CGGCTGTGCG AACGCATTCT GGCATGATGC GATCACCACT CTTGGTTTGC AAGTGTCT 3300 ACACAGCGCT CCGGTTCTTA CGAAAAC TCAAAC TCA GCCACTGAGGTTCT ACCATAAAC 3360 TACACCACCA TTAATTACTA CAAAGACTCC TATGCTGCAC AGCAGGCAAC AGAGTCTC 3420 AAGCAGGATC CAGACAAGTT TGCAAAATCC TGTAAAGACA TATTCACCGA AATGGCAGCG 3480 CCACTGAAGT CCCCATCCGC TGAGGCAATG TGGATACAGT GATCGAGTGG CGCAATTAAC 3540 ATTGGCAACT CCACCATCAC GACGCAAGAA GCGGCTACAT CATAGTCGG CTATGGTGAG 3600 TGGCCTTCCT ACTGCTCAGA TTCTGACGCT ACAGCAGTGG ATAAACCAAC GCGCCCAGAT 3660 GTTTCAGTGA ACAGGTTTTA CATTGGACAC TAAATTGTGG GAGAAATCG TCCAAGGGA 3720 TGGTACTGGA AGTTCCCGGA TGTGTAACTG AAACTGGGGT TTTGGGCAAA ATGCACAAG 3780 TTCCACTACC TCTACCATCA GGTTCTGCAT CCACGTGCAG TGCAATGCCA GTAATTCTC 3840 CACCAAGGAG CACTCCTAGT CGCTGTCCTA CCAGAGTATG TCATTGGGAC AGTGGCAGGC 3900 GGTACAGGGA CGGAAGACAC CCACCCCCCC TACAAGCAGA CCCAACCCGG CGCCGATGGT 3960 TTCGAGTTGC AACACCCGTA CGTGCTTGAT GCTGGCATCC CAATATCACA GTTAACAGTG 4020 TGCCCACACC AGTGGATTAAT TTGAGGACCA ACAATTGTGC TACAATAATG TGCCATACT 4080 attaacgcac tgccttttga ttctgccttg aaccattgca actttggcct gttagttgtg 4140 cctattagcc cactagacta cgaccaagga gcaacgccag taatccctat aactatcaca 4200 ttggccccaa tgtgctctga attcgcaggt cttaggcagg cagtcacgca agggttcccc 4260 accgagctaa aacctggcac aaatcaattt ttaaccaccg atgatggcgt ctcagcacct 4320 attctaccaa acttccaccc caccccgtgt atccacatac ctggtgaagt taggaacttg 4380 ctagagttat gccaggtgga gaccattctg gaggttaaca atgtgcccac gaatgccact 4440 agcttaatgg agagactgcg cttcccggtc tcagcacaag cagggaaagg tgaactgtgt 4500 gcggtgttta gagccgatcc tgggcgaaat ggaccatggc aatccacctt actgggccag 4560 ttgtgcgggt actacaccca atggtcaggg tcattggaag tcaccttcat gtttactgga 4620 tccttcatgg ctaccggcaa gatgctcata gcctatacac cgccaggggg tcctctgccc 4680 aaggaccggg cgaccgccat gttgggcacg cacgtcatct gggattttgg gctgcaatcg 4740 tctgttaccc ttgtaatacc atggatcagt aacactcatt atagagcaca tgcccgagat 4800 ggagtgtttg actattacac tacagggtta gtcagtatat ggtaccagac aaattacgtg 4860 gttccaatcg gtgcgcccaa cacagcctat ataatagcac tagcggcagc ccaaaagaac 4920 ttcactatga aattgtgcaa ggatgctagt gatatcctgc agacgggcac catccaggga 4980 gatagggtgg cagatgtaat tgaaagttcc ataggagata gcgtgagcag agccctcact 5040 cacgctctac cagcacccac aggccaaaac acacaggtga gcagtcatcg actggataca 5100 ggcaaggttc cagcactcca agctgctgaa attggggcat catcaaatgc tagtgacgag 5160 agcatgattg aaacacgttg tgttcttaac tcgcatagta cagctgagac cactcttgat 5220 agtttcttca gtagggcagg attagttgga gagatagatc tccctcttga gggcacaact 5280 aacccaaatg gttatgccaa ctgggacata gatataacag gttacgcgca aatgcgtaga 5340 aaggtagagc tattcaccta catgcgtttt gatgcagagt tcacttttgt tgcgtgcaca 5400 cccaccgggg aggttgtccc acaattgctc caatatatgt ttgtgccacc tggagcccct 5460 aagccagatt ctagggaatc ccttgcatgg caaaccgcca ccaacccctc agtttttgtc 5520 aagctgtcag accctccggc gcaggtttca gtgccattca tgtcacctgc gagtgcttat 5580 caatggtttt atgacggata tcccacattc ggagaacaca aacaggagaa agaccttgaa 5640 tacggggcat gtcctaataa catgatgggt acattctcag tgcggactgt ggggacctcc 5700 aagtccaagt accctttagt ggttaggatt tacatgagaa tgaagcacgt cagggcgtgg 5760 atacctcgcc cgatgcgcaa ccagaactac ctgttcaaag ccaacccaaa ttatgctggc 5820 aactctatta agccaactgg tgccagtcgc acagcgatca ccactcttgg gaaatttgga 5880 caacagtctg gggctattta tgtgggcaac tttagagtgg tcaaccgaca tcttgccacc 5940 cataatgatt gggcaaatct tgtttgggaa gacagctctc gcgacttgct cgtgtcatcc 6000 accactgccc aaggttgtga cacgattgcc cgttgcgatt gccagacagg ggtgtactac 6060 tgtaactcga tgagaaaaca ctacccagtc agtttttcaa aacccagcct gatctatgta 6120 gaggctagcg agtattaccc agccaggtac caatcacatc tcatgctcgc acagggtcac 6180 tcggaacctg gtgattgcgg tggtatcctt aggtgccaac atggcgtcat cggcatagtg 6240 tctactggtg gcaatgggct cgttggcttt gcagacgtca gagacctctt gtggttagat 6300 gaagaagcta tggaacaggg cgtgtccgac tacattaagg gtctcggaga tgcttttgga 6360 acaggcttca ctgacgcagt ctcaagggag gttgaagctc tcaagaacta tcttataggg 6420 tctgaaggag cagttgagaa aattttgaaa aatcttatta aactaatctc tgcactggtg 6480 attgtgatca gaagtgatta cgacatggtt accctcactg caaccttagc gctgataggt 6540 tgtcatggca gtccttgggc ttggattaaa gccaaaacag cctccatctt aggtatccct 6600 atcgcccaaa agcagagcgc ttcctggctc aagaagttca atgacatggc caacgccgct 6660 aaggggttag agtgggtttc caacaagatc agcaaattta ttgattggct taaggagaaa 6720 atagtaccag cagccaggga gaaggttgaa ttcctaaata acttgaaaca gctgccactg 6780 ctagagaatc agatctcgaa cttggaacaa tctgctgctt cacaagagga ccttgaagtc 6840 atgtttggga atgtgtcgta cctagctcac ttctgtcgca agtttcaacc gctatacgcc 6900 acggaagcta aaagagtcta tgccctggag aagagaatga ataactatat gcagttcaag 6960 agcaaacacc gaattgaacc tgtatgtctc attattaggg gctcaccagg caccgggaag 7020 tctctagcca ctggtattat tgctcgagca atcgctgata agtaccactc cagcgtgtac 7080 tcgctcccac cagacccgga tcattttgac ggttacaagc aacaggtggt tacagtgatg 7140 gatgatttgt gtcaaaaccc cgatggtaag gatatgtcct tattctgtca aatggtatcc 7200 accgtagatt tcattccacc aatggcttct ctcgaggaga agggagtttc cttcacctct 7260 aagtttgtca tcgcatccac taatgccagt aatatcatag taccaacagt gtctgattct 7320 gacgctattc gccgcaggtt ctacatggac tgtgacattg aagtgacaga ctcgtacaaa 7380 acagatctag gtagactgga tgcagggcga gccgctaaac tgtgttctga aaataacact 7440 gcaaatttca aacgttgcag cccattagtg tgtgggaaag ccatccaact tagagataga 7500 aagtctaaag tcagatacag tgtggatacg gtggtttcag aacttattag ggaatacagc 7560 aataggtccg ccattggtaa cacaatcgag gctcttttcc aaggtccacc caagttcagg 7620 ccaattagga ttagccttga agaaaaacca gccccagacg ctattagcga tctccttgct 7680 agtgtagata gtgaagaagt gcgccagtac tgcagggatc aaggctggat tattcctgaa 7740 gctcccacca atgtggagcg gcaccttaat agagcggtgc tcgtcatgca atccatcacc 7800 acagtagtgg cggttgtttc gttggtgtac gtcatctaca agctctttgc agggtttcag 7860 ggtgcatatt ctggtgctcc taagcaagtg cttaagaaac ctgctcttcg cacagcaaca 7920 gtgcagggtc cgagccttga ctttgctctc tccctactga gaaggaacat caggcaggtc 7980 caaacagacc aagggcattt caccatgttg ggtgttaggg atcgcttagc agtcctccca 8040 cgccactcac aacctggcaa aaccatttgg attgagcaca aactcgtgaa cgtccttgat 8100 gcagttgaac tggtggatga gcaaggagtc aacctggaat taaccctcat cactcttgac 8160 accaacgaga agtttaggga tatcaccaaa ttcatcccag aaaatatcag cactgctagc 8220 gatgccaccc tagtgatcaa cacggagcac atgccgtcaa tgtttgtccc ggtgggtgac 8280 gttgtgcagt atggcttttt gaatctcagt ggcaagccta cccatcgcac catgatgtac 8340 aattttccta ctaaagcagg acagtgtgga ggagtggtga catctgttgg gaaggttgtc 8400 ggtattcaca ttggtggcaa tggcagacaa ggtttttgcg caggcctcaa aaggagttac 8460 tttgctagtg aacaaggaga gatccagtgg gttaagccca ataaagaaac tggaagactc 8520 aacatcaatg gaccaacccg caccaagtta gaacctagtg tattccatga catcttcgag 8580 ggaaataagg aaccagctgt cttgcacagt aaagaccccc gacttgaggt agattttgaa 8640 caggccctgt tctctaagta tgtgggaaac acactacatg agcctgacga gtacatcaaa 8700 gaggcagctc tacattatgc aaaccaatta aagcaactag aaatcaatac ctctcaaatg 8760 agcatggagg aggcctgcta tggtactgag aatcttgagg ctattgatct tcacactagt 8820 gcaggttacc cctatagtgc cctagggata aagaaaagag acatcttaga ccctaccacc 8880 agggacgtga gtagaatgaa gttctacatg gacaagtatg gtcttgatct tccctactcc 8940 acttatgtca aggacgagct acgctcgatt gataaaatca agaaagggaa gtcccgcctg 9000 atcgaggcca gtagtctaaa tgattcagtg tacctcagaa tggctttcgg gcatttgtat 9060 gaggctttcc acgcaaatcc tgggacgata actggatcgg ccgtggggtg taaccctgac 9120 acattctgga gcaagctgcc aattttgctc cctggttcac tctttgcctt tgactactca 9180 ggctatgatg ccagccttag ccctgtctgg ttcagagcat tagaattggt tcttagggag 9240 atagggtata gtgaagaggc aatctcactc attgagggaa tcaaccacac acatcatgtg 9300 tatcgtaata agacctattg cgtgcttggt gggatgccct caggctgttc aggaacatcc 9360 atcttcaact caatgatcaa caacattatt atcagagcac tgctcataaa aacatttaag 9420 ggcattgatt tggatgaact caacatggtc gcttatggag acgatgtgct cgctagctat 9480 cccttcccaa ttgattgctt ggaactagca aagactggta aggagtatgg tctgaccatg 9540 acccctgctg ataaatctcc ttgctttaat gaggtcaatt ggggtaatgc gaccttcctc 9600 aaaaggggct ttttgcccga tgaacagttt ccatttttga ttcaccctac tatgccaatg 9660 agggagatcc atgagtccat tcgatggacc aaggacgcac ggaacactca agatcatgtg 9720 cggtccttgt gcctcctagc atggcataat ggtaagcaag aatacgagaa gtttgtgagc 9780 acaattaggt ctgtcccagt agggagagcg ttggctattc caaattatga aaatcttaga 9840 CGAAATTGGC TCGAGTTATT TT TAGAGGTTA TACACACCTC AACCCCACCA GAAATCTGGT 9900 CGTGAATGTG ACTGGTGGGG TAAATTTGTT ATAACCAAGA ATAGCAA AAA AAAAAAAA 9960 AAAAAAAAAA AAAAAAAGCT TA 9982 <210> 6 <211> 10187 <212> DNA <213> Artificial <400> 6 GCTAGCGGAG TGTAATACTG GCTTACTATG TTGGC ACTGAT GAGGGTGTC AGTGAAGTGCT 60 TTCATGTGGC AGGAGAAAAA AGGCTGCACC GGTGC GTCAGC AGAATATGTG ATACAGGATA 120 TATTCCGCTT CCTCGCTCAC TGACTCGTAC GCTCGGTCGT TC GACTGCGGCG AGC GGA A 180 ATGGCTTACG AACGGGGCGG AGATTTCC TGGAAGATGC CAGGAAGATACTTAACAGGGA A 240 GTGAGAGGGC CCGGGCAAAG CC GTTTTCC ATAGGCTCCG CCCCCCTGAC AAGCATCACG 300 AAATCTGACG CTCAATCAGT GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGC GT 360 TTCCCCTGGC GGCTCCCTCG TGC GCTCTCCT GTTCCTGCCT TTCGGTTTAC CGGTGT CAT 420 TCCGCTGTTA TGGCCGC GTT GTCTCATT CCACGCCTGA CT CAGTTCCGGG TAGGCAG 480 ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 540 ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 600 ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 660 gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 720 gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 780 gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaaggg gtctgacgct 840 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 900 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 960 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 1020 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 1080 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 1140 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 1200 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 1260 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 1320 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 1380 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 1440 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 1500 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 1560 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 1620 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 1680 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 1740 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 1800 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 1860 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 1920 AAATAGGGGTTCCGCGCACATTTCCC CGAAAAGTGCCCTGACGTGTCGACGC 1980 CCTAATACTGACTC ACTAT AGGT TAAAAC AGCCTGTGGG TTGCACCAC TCA CAGGGCC 2040 TACTGGGC GC AAGC ACTCTG GTACCTCGGT ACCTTTGTGC GCCTGTTTAT ACCCCCCCCC 2100 CCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGT 2160 CTTGATCAAGC ACTTCTGTTTCCCCGGACTGAGTATCAAT AGACTGCTCGC GC GTTGA A 2220 GGAGAAAACGTTCGTTATCCGGCTA ACTACTTCGGAAAACCTAGTAACACCATGAAAGTT 2280 GC GGAGAGCTTCGTT CAGCACTCCCCCAGTG TAGATCAGGTCGATGAGTC ACCGC GTTCC 2340 CCACGGGC GACC GTGGCGGT GGCTGC GTTGGCGGCCTGCCCATGGGGTAACCCATGGGGC 2400 GCTCTAATACTGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCC TCCGGCCCCT 2460 GAA TGC GGCTAATCCCAACT GC GGAGC ACACCCCACAAGCC AGC GGGT AGT GTGTCGTA 2520 ACGGGTAAC TCTGCAGCGGA ACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTAT 2580 ATTGGCTGCTTATGGTGACAATTAAGAATGTTACCATA TAGCTATTGGAT TAGCCATC 2640 CGGTGTGCAA CAGAGCAATT ATTTACCTAT TTATTGGTTT GTACCATTA ACC TC GAATT 2700 CTGTGACCAC CCTTAATTAT ATCTTGACCC TTAACACAGC TAAACC ATAT GATGGTGAGC 2760 AAGGGCGAGG AGCTGTTCAC CGGGGTGGTG CCCATCCTGG TCGAGCTGGA CGGC GACGTA 2820 AACGGCCACA AGTTCAGCGT GTCCGGCGAG GGC GAGGGCGAT GCCACCTAC GGCAAGCTG 2880 ACCCTGAAGT T CATCTGCACC ACCGGCAAGC TGCCC GTCCCTGGCCC ACCCTCGTGACC 2940 ACCCTGACCT ACGGCGTGCA GTGCTTCAGC CGCTACCCCG ACCACATGAAG CAGCACGAC 3000 TTCTTCAAGT CCGCCATGCC C GAAGGCTAC GTCCAGGAGC GCACC ATCTTCTTCAAGGAC 3060 GACGGCAACT ACAAGACCCG CGCCGAGGTG AAGTTCGAGG GC GACACCCT GGTGAACCGC 3120 ATCGAGCTGA AGGGCATCGA CTTCAAGGAG GACGGCAACA TCCTGGGGCA CAAGCTGGAG 3180 TACAAC TACAACAGCC ACAACGTCT ATATCATGGCC GACAAGCAGAAG AACGGCATCAAG 3240 GTGAAC TTC AAGATCCGCC ACAACATCGA GGACGGCAGCG TGCAGCTCGC CGACCAC TAC 3300 CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCGACAACCAC TACCTGAGC 3360 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 3420 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagat gcatgcgatc 3480 accactcttg gttcgcaagt gtctacacag cgctccggtt cttacgaaaa ctcaaactca 3540 gccactgagg gttctaccat aaactacacc accattaatt actacaaaga ctcctatgct 3600 gccacagcag gcaaacagag tctcaagcag gatccagaca agtttgcaaa tcctgttaaa 3660 gacatattca ccgaaatggc agcgccactg aagtccccat ccgctgaggc atgtggatac 3720 agtgatcgag tggcgcaatt aactattggc aactccacca tcacgacgca agaagcggct 3780 aacatcatag tcggctatgg tgagtggcct tcctactgct cagattctga cgctacagca 3840 gtggataaac caacgcgccc ggatgtttca gtgaacaggt tttacacatt ggacactaaa 3900 ttgtgggaga aatcgtccaa gggatggtac tggaagttcc cggatgtgtt aactgaaact 3960 ggggtttttg ggcaaaatgc acaattccac tacctctacc gatcagggtt ctgcatccac 4020 gtgcagtgca atgccagtaa attccaccaa ggagcactcc tagtcgctgt cctaccagag 4080 TATGTCATGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACC CCCCTACAAG 4140 CAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGC 4200 ATCCCAATATCACAGTCAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAAT 4260 TGTGCTACAATAATAGTGCCATACATTAACGCCTCTTCTGATTCTGCCTTGAACCAT 4320 TGCACTTTGGCCTGTTAGTTGTGCCTATTAGCCCAC TAGACTACGACC AAGGAGCAACG 4380 CCAGTAATCCCTATAACTATCACATTGGCCCCAA TGTGCTCTGAATTCGCAGGTCTTAGG 4440 CAGGCAGTCACGCAAGGGTCCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTAACC 4500 ACC GATGATGGC GTCTCAGCACC TATTCTACCAA CTTCCACCCCACCCC GTGTATCCAC 4560 ATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCAT TCTGGAGGTT 4620 AACAATGTGCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCC GGTCTCAGCA 4680 CAAGCAGGGAAAGGTGAAC TGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCA 4740 TGGCAATCCACCTTACTGGGCCAGTTGTGCGG GTACTACACCCCAATGGTCAGGGTCATTG 4800 gaagtcacct tcatgtttac tggatccttc atggctaccg gcaagatgct catagcctat 4860 acaccgccag ggggtcctct gcccaaggac cgggcgaccg ccatgttggg cacgcacgtc 4920 atctgggatt ttgggctgca atcgtctgtt acccttgtaa taccatggat cagtaacact 4980 cattatagag cacatgcccg agatggagtg tttgactatt acactacagg gttagtcagt 5040 atatggtacc agacaaatta cgtggttcca atcggtgcgc ccaacacagc ctatataata 5100 gcactagcgg cagcccaaaa gaacttcact atgaaattgt gcaaggatgc tagtgatatc 5160 ctgcagacgg gcaccatcca gggagatagg gtggcagatg taattgaaag ttccatagga 5220 gatagcgtga gcagagccct cactcacgct ctaccagcac ccacaggcca aaacacacag 5280 gtgagcagtc atcgactgga tacaggcaag gttccagcac tccaagctgc tgaaattggg 5340 gcatcatcaa atgctagtga cgagagcatg attgaaacac gttgtgttct taactcgcat 5400 agtacagctg agaccactct tgatagtttc ttcagtaggg caggattagt tggagagata 5460 gatctccctc ttgagggcac aactaaccca aatggttatg ccaactggga catagatata 5520 acaggttacg cgcaaatgcg tagaaaggta gagctattca cctacatgcg ttttgatgca 5580 gagttcactt ttgttgcgtg cacacccacc ggggaggttg tcccacaatt gctccaatat 5640 atgtttgtgc cacctggagc ccctaagcca gattctaggg aatcccttgc atggcaaacc 5700 gccaccaacc cctcagtttt tgtcaagctg tcagaccctc cggcgcaggt ttcagtgcca 5760 ttcatgtcac ctgcgagtgc ttatcaatgg ttttatgacg gatatcccac attcggagaa 5820 cacaaacagg agaaagacct tgaatacggg gcatgtccta ataacatgat gggtacattc 5880 tcagtgcgga ctgtggggac ctccaagtcc aagtaccctt tagtggttag gatttacatg 5940 agaatgaagc acgtcagggc gtggatacct cgcccgatgc gcaaccagaa ctacctgttc 6000 aaagccaacc caaattatgc tggcaactct attaagccaa ctggtgccag tcgcacagcg 6060 atcaccactc ttgggaaatt tggacaacag tctggggcta tttatgtggg caactttaga 6120 gtggtcaacc gacatcttgc cacccataat gattgggcaa atcttgtttg ggaagacagc 6180 tctcgcgact tgctcgtgtc atccaccact gcccaaggtt gtgacacgat tgcccgttgc 6240 gattgccaga caggggtgta ctactgtaac tcgatgagaa aacactaccc agtcagtttt 6300 tcaaaaccca gcctgatcta tgtagaggct agcgagtatt acccagccag gtaccaatca 6360 catctcatgc tcgcacaggg tcactcggaa cctggtgatt gcggtggtat ccttaggtgc 6420 caacatggcg tcatcggcat agtgtctact ggtggcaatg ggctcgttgg ctttgcagac 6480 gtcagagacc tcttgtggtt agatgaagaa gctatggaac agggcgtgtc cgactacatt 6540 aagggtctcg gagatgcttt tggaacaggc ttcactgacg cagtctcaag ggaggttgaa 6600 gctctcaaga actatcttat agggtctgaa ggagcagttg agaaaatttt gaaaaatctt 6660 attaaactaa tctctgcact ggtgattgtg atcagaagtg attacgacat ggttaccctc 6720 actgcaacct tagcgctgat aggttgtcat ggcagtcctt gggcttggat taaagccaaa 6780 acagcctcca tcttaggtat ccctatcgcc caaaagcaga gcgcttcctg gctcaagaag 6840 ttcaatgaca tggccaacgc cgctaagggg ttagagtggg tttccaacaa gatcagcaaa 6900 tttattgatt ggcttaagga gaaaatagta ccagcagcca gggagaaggt tgaattccta 6960 aataacttga aacagctgcc actgctagag aatcagatct cgaacttgga acaatctgct 7020 gcttcacaag aggaccttga agtcatgttt gggaatgtgt cgtacctagc tcacttctgt 7080 cgcaagtttc aaccgctata cgccacggaa gctaaaagag tctatgccct ggagaagaga 7140 atgaataact atatgcagtt caagagcaaa caccgaattg aacctgtatg tctcattatt 7200 aggggctcac caggcaccgg gaagtctcta gccactggta ttattgctcg agcaatcgct 7260 gataagtacc actccagcgt gtactcgctc ccaccagacc ggatcattt tgacggttac 7320 aagcaacagg tggttacagt gatggatgat ttgtgtcaaa accccgatgg taaggatatg 7380 tccttattct gtcaaatggt atccaccgta gatttcattc caccaatggc ttctctcgag 7440 gagaagggag tttccttcac ctctaagttt gtcatcgcat ccactaatgc cagtaatatc 7500 atagtaccaa cagtgtctga ttctgacgct attcgccgca ggttctacat ggactgtgac 7560 attgaagtga cagactcgta caaaacagat ctaggtagac tggatgcagg gcgagccgct 7620 aaactgtgtt ctgaaaataa cactgcaaat ttcaaacgtt gcagcccatt agtgtgtggg 7680 AAAGCCATCC AACTTAGAGA TAGAAAGTCT AAAGTCAGAT ACAGTGTGGA TACGGTGGTT 7740 TCAGAAGTGA TTTGGGAAAT ACGCAATAGG TCCGCCATCG GCAACACGAT GAGGCTCTT 7800 TTCCAAGGTC ACACCAAGTT CAGGCCAATT AGGATTAGCC TTGAAGAAAA ACCAGCCCCA 7860 GACGCTATTA GCATCTCCTT GCTAGTGTAG ATAGTGAAGA AGTGCGCCAG TACTGCAGG 7920 GATCAAGGCT GGATTATTC CTGAAGCTCC CACCAATGTG GAGCGGCACCT TAATAGAGC 7980 GTGCTCGTCA TGCAATCCAT CACCACAGTA GTGGCGGTTG TTTCGTTGGT GTACGTGATC 8040 TACAAGCTCT TTGCAGGGTT TCAGGGTGCA TATTCTGGTG CTCCTAAGCA AGTGCTTAAG 8100 AAACCTGCTC TTCGCACAGC AACAGTGAGG GTCCGAGCCT TGACTTTGCT CTCTCCCTA 8160 CTGAGAAGGA ACATCAGGCA GGTCCAAACA GACCAAGGGC ATTTCCACAT GTTGGGTGTT 8220 AGGGATCGCT TAGCAGTCCT CCCACGCCAC TCACAACCTG GCAAAACCAT TTGGATTGAG 8280 CACAAACTCG TGAACGTCCT TGATGCAGTT GAACCGGTGG ATGAGCAAGG AGTCAACCTG 8340 GAATTAACCC TCAACACTCT TTGACACCAA CGAGAAGTTT AGGGATATCA CCAAATTCAT 8400 ccagaaaata tcagcactgc tagcgatgcc accctagtga tcaacacgga gcacatgccg 8460 tcaatgtttg tcccggtggg tgacgttgtg cagtatggct ttttgaatct cagtggcaag 8520 cctacccatc gcaccatgat gtacaatttt cctactaaag caggacagtg tggaggagtg 8580 gtgacatctg ttgggaaggt tgtcggtatt cacattggtg gcaatggcag acaaggtttt 8640 tgcgcaggcc tcaaaaggag ttactttgct agtgaacaag gagagatcca gtgggttaag 8700 cccaataaag aaactggaag actcaacatc aatggaccaa cccgcaccaa gttagaacct 8760 agtgtattcc atgacatctt cgagggaaat aaggaaccag ctgtcttgca cagtaaagac 8820 ccccgacttg aggtagattt tgaacaggcc ctgttctcta agtatgtggg aaacacacta 8880 catgagcctg acgagtacat caaagaggca gctctacatt atgcaaacca attaaagcaa 8940 ctagaaatca atacctctca aatgagcatg gaggaggcct gctatggtac tgagaatctt 9000 gaggctattg atcttcacac tagtgcaggt tacccctata gtgccctagg gataaagaaa 9060 agagacatct tagaccctac caccagggac gtgagtagaa tgaagttcta catggacaag 9120 TATGGTCTTGA TCTTCCCTAC TCCACTTATG TCAAGGACGA GCTACGCTCG ATTGATAAA 9180 ATCAAGAAAG GGAAGTCCCC CTGATCGAGG CCAGTAGTCT AAATGATTC AGTGTA CCTC 9240 AGAATGGCTT TCGGGCATTT GTATGAGGCT TTCCACGCAA ATCCTGGGAC GATAACTGGA 9300 TCGGCCGTGG GGTGTAACCC TGACACATTC TGGAGCAAGC TGCCAATTTT GCTCCCTGGT 9360 TCACTCTTTG CCTTTGACTA CTCAGGCTAT GATGCCAGCC TTAGCCCTGT CTGgttcaga 9420 GCATTAGAAT TGgttcttag ggagataggg tatagtgaag aggcaatctc actcattgag 9480 GGAATCAACC ACACACATCA TGTGTATCGT AATAAGACCT ATTGCgtgct tggtgggatg 9540 CCCTCAGGCT GTTCAGGAAC ATCCATCTTC AACTCAATGA TCAACAACAT TATTATCAGA 9600 GCACTGCTCA TAAAAACATT TAAGGGCATT GATTTGGATG AACTCAACAT Ggtcgcttat 9660 GGAGACGATG TGCTCGCTAG CTATCCCTTC ccaattgatt gcttggaact agcaaagact 9720 GGTAAGGAAT ATGgtctgac catgacccct gctgataaat ctccttgctt taatgaggtc 9780 AATTGGGGTA ATGCGACCTT CCTCAAAAGG GGCTTTTTGC CCGATGAACA GTTCCATTT 9840 ttgattcacc ctactatgcc aatgagggag atccatgagt ccattcgatg gaccaaggac 9900 gcacggaaca ctcaagatca tgtgcggtcc ttgtgcctcc tagcatggca taatggtaag 9960 caagaatacg agaagtttgt gagcacaatt aggtctgtcc cagtagggag agcgttggct 10020 attccaaatt atgaaaatct tagacgaaat tggctcgagt tattttagag gttatacaca 10080 cctcaacccc accagaaatc tggtcgtgaa tgtgactggt gggggtaaat ttgttataac 10140 cagaatagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcttat 10187 <210> 7 <211> 19 <212> DNA <213> Artificial <400> 7 taatacgact cactatagg 19 <210> 8 <211> 30 <212> DNA <213> Artificial <400> 8 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 30 <210> 9 <211> 39 <212> DNA <213> Artificial <400> 9 gctagcgctt tttttttttt tttttttttt ttttttttt 39 <210> 10 <211> 55 <212> DNA <213> Artificial <400> 10 gacgcggccg ctaatacgac tcactatagg ttaaaacagc ctgtgggttg caccc 55 <210> 11 <211> 22 <212> DNA <213> Artificial <400> 11 gcactgcacg tggatgcaga ac 22 <210> 12 <211> 33 <212> DNA <213> Artificial <400> 12 gacgcggccg cgttctgcat ccacgtgcag tgc 33 <210> 13 <211> 22 <212> DNA <213> Artificial <400> 13 aagtcgcgag agctgtcttc cc 22 <210> 14 <211> 33 <212> DNA <213> Artificial <400> 14 gacgcggccg cgggaagaca gctctcgcga ctt 33 <210> 15 <211> 28 <212> DNA <213> Artificial <400> 15 aattgtacat catggtgcga tgggtagg 28 <210> 16 <211> 39 <212> DNA <213> Artificial <400> 16 gacgcggccg ccctacccat cgcaccatga tgtacaatt 39 <210> 17 <211> 73 <212> DNA <213> Artificial <400> 17 gctagcgctt tttttttttt tttttttttt tttttttttg ctattctggt tataacaaat 60 ttacccccac cag 73 <210> 18 <211> 18 <212> DNA <213> Artificial <400> 18 cctgacgtgt cgacgcgg 18 <210> 19 <211> 49 <212> DNA <213> Artificial <400> 19 cctcgccctt gctcaccatc atatggttta gctgtgttaa gggtcaaga 49 <210> 20 <211> 49 <212> DNA <213> Artificial <400> 20 tcttgaccct taacacagct aaaccatatg atggtgagca agggcgagg 49 <210> 21 <211> 66 <212> DNA <213> Artificial <400> 21 CGCTGTGTAG ACACCTTGCG A ACCAAGAGTG GTGATCGCAT GCACTTGTAC AGCTCGTCC 60 ATGCCG 66 <210> 22 <211> 66 <212> DNA <213> Artificial <400> 22 Cggcatggac gagctgtaca agatgcatgc gatcaccact cttggttcgc aagtgtctac 60 ACAGCG 66 <210> 23 <211> 21 <212> DNA <213> Artificial <400> 23 CTGCACGTGG ATGCAGAACC C 21 <210> 24 <211> 21 <212> DNA <213> Artificial <400> 24 CTGCACGTGG ATGCAGAACC C 21 <210> 25 <211> 53 <212> DNA <213> Artificial <400> 25 GAAATCTTCG AGTGTGAAGA CCATTCTAGA GTT TAGCTGT GTTAAGGGTC AAG 53 <210> 26 <211> 53 <212> DNA <213> Artificial <400> 26 CTTGACCCTT AACACAGCTA AACTCTAGAA TG GTCTTCAC ACTCGAAGAT TTC 53 <210> 27 <211> 27 <212> DNA <213> Artificial <400> 27 cgcatgcatc gccagaatgc gttcgca 27

Claims

1. A cDNA, characterized in that It contains nucleic acid sequence of EV71 strain and nucleic acid sequence of a low copy plasmid backbone; wherein, the nucleic acid sequence of EV71 strain covers EV71 virus 5' to 3' positive sequence, including virus 5' and 3' non-coding region and an open reading frame encoding virus protein; The amino acid sequence of the virus protein open reading frame is shown as SEQ ID NO 4. The coding sequence of the low copy plasmid backbone is shown as SEQ ID NO 3. The nucleic acid sequence of the EV71 strain is shown as SEQ ID NO 2. The cDNA, whose sequence is shown as SEQ ID NO 1, further includes the sequence of reporter luciferase or fluorescent protein inserted in the nucleic acid sequence of EV71 strain.

2. A cDNA, characterized in that, The sequence of reporter luciferase or fluorescent protein is inserted in the nucleic acid sequence of EV71 strain of the cDNA of claim 1.

3. A recombinant virus, wherein, It contains the cDNA of claim 1.

4. A subgenomic replicon, characterized in that, Its cDNA sequence is shown as claim 1.

5. A double-stranded DNA, characterized in that, It can produce the cDNA of claim 1.

6. A plasmid, characterized in that, It contains the double-stranded DNA of claim 4.

7. The plasmid of claim 6, wherein, It can transcribe to produce full-length infectious RNA of EV71 strain.

8. A vaccine comprising a polynucleotide of claim 1. It is prepared from the plasmid of claim 6 or 7.

9. A viral vector, characterized in that, It is prepared from the plasmid of claim 6 or 7.

10. A viral particle, characterized in that, It is produced from the cDNA of claim 1, or prepared from the plasmid of claim 6 or 7.

11. A method for preparing an EV71 virus antibody, characterized in that, It uses the cDNA of claim 1 or the virus particle of claim 10, wherein the virus particle of claim 10 is used to immunize animals and isolate antibodies, or to screen human antibody library.

12. A kit for detecting EV71, characterized in that, It contains the cDNA of claim 1 or the virus particle of claim 10.

13. Use of the cDNA of claim 1 or the virus particle of claim 10 in constructing cell or animal model for screening anti-virus EV71 drugs.

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