CN112094822A - Infectious cDNA clone based on EV71 strain and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and provides a stable infectious cDNA clone based on an EV71 strain which is clinically separated, derivative clones containing various reporter genes, and various mutant clones constructed by taking the infectious cDNA clone as a female parent; and various recombinant viruses, subunit viral particles produced using these clones; and animal models established by infecting animals with various recombinant viruses produced by the clones; and the use of these viral or subunit viral particles for vaccine development and diagnostic reagents; and the use of the virus as a gene therapy vector or an expression vector. The invention provides a new tool and a new way for detection, prevention and immunization of EV71 virus infection, and provides possibility for gene therapy and vaccine development by using the EV71 strain infectious clone as a virus vector.

Description

Infectious cDNA Cloning Based on EV71 Strain and Its Application

technical field

The invention belongs to the field of biomedicine, and in particular relates to the construction of an infectious cDNA clone based on a clinically isolated EV71 strain (js1), a virus produced by using the cDNA clone and its derivative clones, and a virus with a reporter gene and The established animal models are used in antiviral drug development, vaccine development, and virus diagnosis.

Background technique

The prior art discloses that enterovirus is a general term for a class of viruses, including 3 types of poliovirus (Poliovirus), 23 types of Coxsackie virus (Coxsackie virus A), Coxsackie virus (Coxsackie virus A) B) 6 types of viruses, 31 types of ECHO virus, 68-71 types of enterovirus, a total of 67 types. Enteroviruses discovered after traditional typing are named according to the order of discovery. The new types of enteroviruses that have been discovered now include enteroviruses 68, 69, 70, 71 and 72. Novel enterovirus type 71, or EV71 for short, belongs to the Picornaviridae family, the genus Enterovirus, which was isolated from Australia and the United States in 1969, and was isolated in Japan in 1973, and is considered to be the epicenter of the outbreak of hand, foot and mouth disease in children. Major pathogen (Schmidt et al. J Infect Dis 1974, 129:304-309; Hagiwara et al. Intervirology 1978, 9:60-63.). Before 1988, EV71 virus mainly caused outbreaks of hand, foot and mouth disease in infants and young children in the United States, Japan, Europe and Australia (Weng et al. Microbes Infect 2010; 12: 505-10; Tagaya et al. Jpn J Med Sci Biol 1975 ;28:231-4;Blomberg et al.Lancet 1974;2:112;Nagy et al..Arch Virol 1982;71:217-27;Kennett et al.Bull World Health Organ 1974;51:609-15;Gilbert et al . Pediatr Infect Dis J 1988;7:484-8). Since 1990, EV71 virus has caused a series of outbreaks in the Asia-Pacific region (Chanet al. Clin Infect Dis 2000; 31: 678-83; Tu et al. Emerg Infect Dis 2007; 13: 1733-41; Jeong et al. al.. Arch Virol 2010;155:1707-12). As of 2014, EV71 infection has spread to all continents and countries around the world. Studies have shown that EV71 infection is mainly distributed in the Asia-Pacific region, and EV71 infection is also distributed in North America, South America, Europe and Australia.

The study reported that EV71 virus is a single-stranded positive-stranded RNA virus whose genome can encode a single-length open reading frame (ORF), and also contains two long non-coding regions 5'TURs and 3'TURs in two segments of the genome. 5'TURs contain internal ribosome entry sites (IRES) that initiate viral translation (Hellen et al. Genes Dev. 2001; 15, 1593–1612). After translation, the virus-encoded open reading frame is cleaved and processed into a single viral protein by protease encoded by the virus itself, including the structural proteins VP4, VP2, VP3, VP1 that make up the virus particle and the non-structural proteins 2A, 2B, 2C responsible for viral replication, 3A, 3B, 3C and 3D (Racaniello, et al. FieldsVirology. 2007, Fifth edition).

Studies have reported that primates can be used as an infection model for EV71. As early as 1978, Hashimoto et al. reported that 1.8-3.8kg cynomolgus monkeys could be infected with the EV71 virus strain isolated from the fecal specimen of a 3-year-old child after 9 weeks of isolation. Neurotoxicity, cynomolgus monkeys showed clinical symptoms of nerve damage on the fourth day of infection, and the degree of damage was positively correlated with virus titer. And EV71 virus can induce 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 with symptoms such as brain infection, pulmonary edema, and hemorrhage accompanied by nerve damage. Intravenous and respiratory system infections can directly lead to nervous system infections. Therefore, models for different research purposes can be obtained through different infection routes (Zhang et al. Lab Invest. 2011;91:1337-50). In addition, there are rhesus monkey animal models that can cause central nervous system diseases, (Liu et al. Virology. 2011; 412:91-100).

Non-primate models of EV71 have also been reported, for example, the mouse adaptive mutant EV71 strain EV71/MP4 can infect ICR mice with neurological and lung 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 severely diabetic mice (NOD/SCID mice) to passage the virus to obtain a mouse-adapted EV71 strain that can infect 3-week-old NOD/SCID mice. This mouse model is a natural killer cell Function is suppressed and functional T and B cells are absent. Moreover, the obtained murine-adapted strain mainly infects the central nervous system, heart and skeletal muscle of animals (Arita et al. J Virol. 2008, 82(4): 1787-97). Using AG129 mice, immunodeficient mice deficient in interferon receptor alpha, beta and gamma, AG129 mice 2 weeks of age or younger can be infected with the natural strain of EV71 and show symptoms of 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 EV71Isehara/Japan/99 (Isehara) strain; studies have shown that the construction of EV71 mouse models requires special mouse-adapted strains or genetically deleted or modified mice .

The study also reported that the genomic RNA of a single positive-strand RNA virus is released and can be directly translated as an mRNA template after entering the cytoplasm of the host cell; the viral non-structural proteins generated by translation recruit the viral genome to form the initiation of a replication complex. The gene replication and life cycle of the virus, so the genomic RNA of the single positive-stranded RNA virus is infectious, and after being introduced into the host cell, the entire life cycle of the virus can be completely initiated (Racaniello, et al. Science. 1981, 214 (4523) :916). The method for constructing infectious clones usually uses the total RNA of virus-infected cells as a template, reverse-transcribes it into complementary DNA (cDNA), and then clones the viral fragments into a cloning vector to form an infectious clone of the virus. The constructed infectious clones utilize in vitro transcription to generate complete viral RNA, and then transfect the viral RNA into host cells to initiate the viral life cycle and produce progeny viruses. Alternatively, if the constructed infectious clone has 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 viral life cycle and producing progeny viruses.

Mouse model studies have confirmed that glutamate at position 145 of EV71 is the main site of virus-induced mouse death, and methylation of lysine at position 149 of VP2 can synergistically promote the ability of VP1 145E to cause mouse death (Huang et al.Virology.2012 , 422(1):132-43). This viral site is easily mutated to 145G during the in vitro passage of the virus, resulting in a decrease in the ability of the virus to infect animals (Yi et al. Unpublished data).

Based on the basis and status of the prior art, the inventors of the present application intend to provide an infectious cDNA clone based on EV71 strain and its application.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an infectious cDNA clone based on EV71 strain and its application based on the basis and current state of the prior art. Specifically, it relates to an infectious cDNA clone of a stable EV71 strain, and the clone and its derived viral RNA can self-replicate in cells, produce progeny virus particles and express reporter genes.

Another technical problem to be solved by the present invention is to provide recombinant viruses or subunit virus particles, plasmids, etc. constructed on the basis of the above clones, so as to provide support for the construction of animal models, the development of vaccines and the development of antiviral drugs.

The present invention isolates an EV71 strain (named js1) from the clinic, which does not require mouse adaptive mutation and can infect mice without genetic background changes, and can produce a virus with stable gene sequence by constructing its infectious clone particles, infect ordinary mice, and establish a simple and efficient EV71 animal infection model.

more specific,

The present invention provides a cDNA comprising the nucleic acid sequence of the 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' forward polar sequence of the EV71 virus, including the virus 5' and 3' non-coding regions and an open reading frame encoding a viral protein.

Preferably, it also includes the sequence of reporter gene luciferase or fluorescent protein inserted into the nucleic acid sequence of EV71 strain.

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

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

The nucleic acid sequence of the EV71 strain is shown in SEQ ID NO 2.

In a preferred embodiment of the present invention, the infectious cDNA clone of the EV71 strain has the sequence shown in SEQ ID NO 1.

In one embodiment of the present invention, the construction of a stable infectious cDNA clone of a clinically isolated EV71 strain (nucleic acid sequence 1) and its derivative clones containing various reporter genes (nucleic acid sequence 5, Nucleic acid sequence 6), and various mutant clones constructed therefrom. The viral RNA produced by these clones can self-replicate in cells, produce progeny virus particles and express reporter genes.

The present invention also includes recombinant virus clones containing heterologous reporter sequences or target genes constructed by replacing Nluc or EGFP with the sequences described in nucleic acid sequence 6 or nucleic acid sequence 7 as the parent and their sequences.

The present invention also includes various chimeric virus infectious clones and various chimeric viruses produced by recombinant virus clones containing heterologous reporter sequences or genes of interest, and various viral particles containing reporter genes or foreign genes.

The present invention also includes recombinant virus clones constructed from full-length infectious clone sequences and inserted into the viral protein with a heterologous resistance sequence in the same open reading frame and their sequences.

Specifically, the present invention provides an infectious cDNA clone (nucleic acid sequence 1) comprising a clinically isolated EV71 strain (js1), the infectious clone (nucleic acid sequence 1) comprising a full-length EV71 strain (js1) nucleotide sequence (nucleotide sequence 2) and a low-copy plasmid backbone (nucleotide sequence 3). Nucleic acid sequence 2 covers the 5' to 3' positive-sense sequence of the EV71 virus, which includes the 5' and 3' non-coding regions of the virus and an open reading frame (open reading frame) encoding a viral protein, open reading Frame virus encoding protein (protein sequence 4), insert reporter gene luciferase NanoLuc (Nluc) and fluorescent protein EGFP into this infectious clone (nucleic acid sequence 1), respectively constitute an infectious clone with Nluc (nucleic acid sequence 5) And infectious clones with EGFP (nucleic acid sequence 6), and mutant virus clones (adapted virus), attenuated virus clones (live-attenuated virus), replication-defective virus clones obtained by means of changing nucleic acid based on these clones Derivatives such as defective virus and replication-competent non-infectious virus, such as subgenomic replicons including deletion of structural proteins.

The above sequences 1-6 are as follows:

Nucleic acid sequence 1, SEQ ID NO 1:

GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCAC CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTAT AGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACATGGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTA ACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCAT GGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCC GGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATA GGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTT CAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAAC CCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACC ATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat

Nucleic acid sequence 2, SEQ ID NO 2:

TTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACATGGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACT ATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGC AATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGC GCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGT TGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAG AACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCG CACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATG ACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGC

Nucleic acid sequence 3, SEQ ID NO 3:

AGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGC

Protein sequence 4, SEQ ID NO 4:

MGSQVSTQRSGSYENSNSATEGSTINYTTINYYKDSYAATAGKQSLKQDPDKFANPVKDIFTEMAAPLKSPSAEACGYSDRVAQLTIGNSTITTQEAANIIVGYGEWPSYCSDSDATAVDKPTRPDVSVNRFYTLDTKLWEKSSKGWYWKFPDVLTETGVFGQNAQFHYLYRSGFCIHVQCNASKFHQGALLVAVLPEYVIGTVAGGTGTEDTHPPYKQTQPGADGFELQHPYVLDAGIPISQLTVCPHQWINLRTNNCATIIVPYINALPFDSALNHCNFGLLVVPISPLDYDQGATPVIPITITLAPMCSEFAGLRQAVTQGFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKGELCAVFRADPGRNGPWQSTLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGGPLPKDRATAMLGTHVIWDFGLQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGAPNTAYIIALAAAQKNFTMKLCKDASDILQTGTIQGDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLEGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTLGKFGQQSGAIYVGNFRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCDCQTGVYYCNSMRKHYPVSFSKPSLIYVEASEYYPARYQSHLMLAQGHSEPGDCGGILRCQHGVIGIVSTGGNGLVGFADVR DLLWLDEEAMEQGVSDYIKGLGDAFGTGFTDAVSREVEALKNYLIGSEGAVEKILKNLIKLISALVIVIRSDYDMVTLTATLALIGCHGSPWAWIKAKTASILGIPIAQKQSASWLKKFNDMANAAKGLEWVSNKISKFIDWLKEKIVPAAREKVEFLNNLKQLPLLENQISNLEQSAASQEDLEVMFGNVSYLAHFCRKFQPLYATEAKRVYALEKRMNNYMQFKSKHRIEPVCLIIRGSPGTGKSLATGIIARAIADKYHSSVYSLPPDPDHFDGYKQQVVTVMDDLCQNPDGKDMSLFCQMVSTVDFIPPMASLEEKGVSFTSKFVIASTNASNIIVPTVSDSDAIRRRFYMDCDIEVTDSYKTDLGRLDAGRAAKLCSENNTANFKRCSPLVCGKAIQLRDRKSKVRYSVDTVVSELIREYSNRSAIGNTIEALFQGPPKFRPIRISLEEKPAPDAISDLLASVDSEEVRQYCRDQGWIIPEAPTNVERHLNRAVLVMQSITTVVAVVSLVYVIYKLFAGFQGAYSGAPKQVLKKPALRTATVQGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQGEIQWVKPNKETGRLNINGPTRTKLEPSVFHDIFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTLHEPDEYIKEAALHYANQLKQLEINTSQMSMEEACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSRMKFYMDKYGLDLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYEAFHANPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYSEEAISLIEGI NHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRALLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSPCFNEVNWGNATFLKRGFLPDEQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPNYENLRRNWLELF

Nucleic acid sequence 5, SEQ ID NO 5:

GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCAC CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTAT AGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACtctagaatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgatgcatGCGATCACCACTCTTGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCC CAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAAT TGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCC ACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGG GCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTT CACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTG ATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat

Nucleic acid sequence 6, SEQ ID NO 6:

GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCAC CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTAT AGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACcatatgATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgac ttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagatgcatGCGATCACCACTCTTGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACC GATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCG AGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTC AAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCT CGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCAT CACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTT GAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCC CAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat.

The present invention also includes the expression products of these cDNA clones.

The present invention also includes double-stranded DNA containing the above cDNA, double stranded DNA capable of producing a full-length infectious clone sequence, positive-sense cDNA or negative-sense cDNA.

The present invention also includes plasmids containing the above-mentioned cDNA or double-stranded DNA.

Preferably, the plasmid can be transcribed to produce the full-length infectious RNA of the EV71 strain or its mutant, can be transcribed in vitro to produce a plasmid containing the full-length infectious RNA of the full-length EV71 strain (js1), and can be produced by in vitro transcription. Plasmids and derivatives containing the full-length infectious RNA of the EV71 strain (js1).

Among them, the derivative plasmids include:

A. The recombinant virus clone obtained by replacing the partial sequence of the full-length infection clone of EV71 strain (js1) in claim 1 with the partial sequence of other isolates;

B. a mutant virus clone obtained by mutating the sequence in the full-length infection clone of EV71 strain (js1) in claim 1 or 2 by gene mutation;

C. Live-attenuated, replication competent non-infectious and defective variants of virus produced by full-length infected clones of EV71 strain (js1) and other derivative clones.

The present invention provides a plasmid containing the above-mentioned double-stranded DNA or its derivatives.

Preferably, it is capable of transcribing the full-length infectious RNA of the EV71 strain or a mutant thereof.

The present invention also provides a vaccine or viral vector, which is prepared according to the above-mentioned plasmid;

The present invention provides a virus particle prepared from the above-mentioned cDNA clone or plasmid;

For example, live-attenuated virus particles, replicationcompetent non-infectious virus particles and non-replicative virus particles (defective variants);

The virus can be isolated and purified to obtain anti-EV71 virus antibodies by immunizing animals, and can also be used to screen human antibody libraries; or, used to prepare kits for detecting EV71 virus and various cell lines, tissues and animal infection models .

The above cell lines, tissues and animal infection models can be used to screen drugs against EV71 virus.

The present invention also includes the detection method and preparation method of the above-mentioned virus vector and virus particle;

For example, the viral particles are used to immunize animals and isolate antibodies, or to screen human antibody libraries.

In another aspect, the present invention provides a kit for detecting EV71, which contains the above-mentioned cDNA or virus particles.

The present invention also provides a method for preparing an antiviral EV71 drug, which uses the above cDNA or virus particles to construct a cell or animal model for screening antiviral EV71 drugs; or, uses the above cDNA or virus particles to construct a cell or animal model , for the screening of antiviral EV71 drugs.

The infectious clone of the present invention (nucleic acid sequence 1) is a complete plasmid consisting of a DNA sequence. It 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). Plasmids are closed double-stranded DNAs that are covalently bonded. It contains a positive-sense strand consistent with the mRNA sequence and a complementary antisense or negative-sense strand.

The full-length nucleic acid sequence (nucleic acid sequence 2) of the EV71 strain (js1) contained in the infectious clone (nucleic acid sequence 1) of the present invention includes the untranslated region (non-translated region) at the 5' end of the viral positive sense sequence. -translated region, NTR), an open reading frame (open reading frame, ORF) and 3' terminal untranslated region (3'-NTR). In this infectious clone, the 5' end of the viral full-length nucleic acid sequence contains a T7 promoter (TAA TAC GAC TCA CTA TAGG, SEQ ID NO 7) (Figure 1A), which can be transcribed in vitro by a commercial T7 transcription kit Full-length viral RNA is transcribed; contains a 30-nucleotide polyA tail (AAAAA AAAAA AAAAA AAAAA AAAAAAAAAA, SEQ ID NO 8) at the 3' end of the viral full-length nucleic acid sequence (FIG. 1A).

RD cells were infected with clinically isolated virus strains. When the cells were cytopathic, the total RNA of the cells was extracted and reverse transcribed using EV71 specific primers (GCT AG CGCT tttttttttt tttttttt ttttttt ttttt, SEQ ID NO 9), and then reverse transcription (Superscript II reverse transcriptase) obtained cDNA was amplified by PCR. The full-length EV71 gene component was amplified in 4 segments (Figure 1A), and the amplification primer was 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 NO11), 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 GCCGC G GGAA 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 the pANCR vector by step-by-step cloning to obtain the final full-length cDNA clone, named pEV71-js1 (Fig. 1A),

After linearization with HindIII in vitro, the infectious clone was transcribed by the T7 transcription kit to contain the full-length viral RNA and its 3'-terminal polyA tail. After the in vitro-produced viral RNA is introduced into host cells such as Vero cells by electroporation or transfection, the viral RNA is used as a translation template to translate its ORF to produce a viral polypeptide (protein sequence 4); the viral polypeptide is processed to form a viral structure Proteins and non-structural proteins start the entire virus life cycle and produce progeny viruses.

Due to degeneracy based on coding, the same functional protein product can still be obtained by changing codons without changing the protein sequence; the present invention includes other nucleic acid sequences and infectious clones encoding the same as "Protein Sequence 4".

The virus produced by the infectious clone (nucleic acid sequence 1) of the present invention exhibits strong replication ability in cells (Fig. 2), and can be used to infect in vitro cultured cell lines, neural tissues, mice (Fig. 6) or Monkeys and others established viral infection cell models and animal infection models for drug research and development.

This infectious clone (nucleic acid sequence 1) was engineered by inserting a reporter gene in a specific region of the virus (before the VP4 protein coding region), the reporter gene being translationally initiated by the IRES of the virus. An additional amino acid site (AITTL) was added to the C-terminus of the reporter gene (Fig. 1B), which was recognized and cleaved by the viral 3C protease, resulting in the normal N-terminus of VP4. We successfully inserted reporter gene luciferase NanoLuc (Nluc) and fluorescent protein EGFP into this infectious clone (nucleic acid sequence 1), which constituted an infectious clone with Nluc (nucleic acid sequence 5) and an infectious clone with EGFP, respectively. clone (nucleic acid sequence 6) (FIG. 1B). Each reporter gene was ligated into the pEV71-js1 plasmid (nucleic acid sequence 1) by fusion PCR and named as pEV71-js1-Nluc (nucleic acid sequence 5) (Fig. 1B) and pEV71-js1-EGFP (nucleic acid sequence 6) (Fig. 1C) ; This infectious clone is the same as above. After being linearized by HindIII in vitro, the full-length viral RNA is transcribed by the T7 transcription kit. The viral life cycle is initiated, producing progeny viruses (Figure 2). The virus expresses 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 by fluorescence microscopy (Figure 4) or detected by flow cytometry. The resulting progeny virus containing the reporter gene segment re-infects new cells, where it can replicate efficiently. Since the reporter gene is in the same open reading frame as the viral protein, its expression level reflects the viral protein level and also the viral replication level. And the recombinant virus containing the reporter gene has no loss of the reporter gene in the continuous passage for a long period of time (Fig. 4). The recombinant virus containing the reporter gene can quickly and conveniently detect the level of virus replication and packaging, and can be used to study the life cycle of the virus, virus-host interaction, virus immunology and the development of antiviral drugs.

This infectious clone (nucleic acid sequence 1) is modified with reference to other viruses belonging to the genus Enterovirus, such as deleting the VP4-VP3-VP2-VP1 region of the structural protein of the virus, which can constitute the subgenomic replicon of the virus and other non-replicating viruses. Infectious virus (replication competent non-infectious), the subgenomic replicon is capable of viral gene replication, but cannot package progeny virus due to the lack of viral structural proteins. At the same time, this subgenomic replicon RNA can be trans-complemented by the expressed structural protein and packaged into recombinant subviral particles (RSPs) (Barclay, et al. J GenVirol. 1998, 79:1725- 1734; Jia, et al. J Virol. 1998, 72: 7972-7977), this subviral particle can carry out a round of infection, but because the genome does not encode a structural protein, it cannot be packaged again after infection. Non-replicating viral particles (defective variants). These non-replicating viral particles can serve as a form of vaccine.

This infectious clone (nucleic acid sequence 1) can be engineered to constitute a live-attenuated virus, which can be used as a vaccine. Referring to the attenuation strategy of Polio virus, which is also a picornaviridae, attenuated vaccine can be constructed by mutation on 5'NTR (Arita, et al. J Virol. 2008, 82: 1787-1797).

The virus produced by the infectious clone infects mice, and establishes a convenient and stable animal infection model. In the present application, it was found that the lethality of the initially isolated virus strain decreased after infecting neonatal mice after more than 3 passages. After passage, the 145 position of VP1 of the virus is mutated from E to G. Therefore, the mouse model established by the infection of the isolated virus has poor consistency. The use of infectious clone to generate virus can ensure that the virus sequence is not affected by cell passage. In this application, neonatal mice of different strains (ICR, Balb/c, C57) were infected with the virus obtained by the above infectious clone, and 100% mortality could be obtained within 9 days (Fig. 6B), but the mice carrying the 145G mutation of VP1 The virus did not lead to death of mice after infection (Fig. 6C). This animal model can be conveniently used for antiviral drugs and vaccine evaluation.

A new type of enterovirus type 71 (Human enterovirus type 71, EV71) is a member of the enterovirus group in the Picornaviridae family of Picornaviridae . EV71 is the main pathogen causing HFMD in children worldwide, and it can cause mild and severe HFMD in children. The virus can infect the central nervous system and cause damage to the central nervous system, but the mechanism is unknown. There are currently no effective antiviral drugs for EV71. In the present invention, a stable virus full-length cDNA clone is constructed by isolating a clinical EV71 strain by molecular cloning, and it is confirmed by in vitro transcription of RNA and transfection of Vero cells that the viral RNA derived from the cDNA clone can produce EV71 virus; Further, the application has constructed a recombinant virus containing reporter gene Gluc (Gaussia luciferase) and EGFP, and confirmed that the recombinant virus containing reporter Gluc and EGFP has the ability to infect host cells and cause cytopathic effects, and utilize the EV71 virus of infectious clone source to infect. The immune-competent ICR, Bab/C and C57 suckling mice caused 100% of the infected mice to develop neurological damage and die within 10 days.

The present invention provides stable, infectious cDNA clones based on a clinically isolated EV71 strain, its derivative clones containing various reporter genes, and various mutant clones constructed therefrom; and the use of these clones to generate Various recombinant viruses and subunit virus particles produced by these clones; and animal models established by infecting animals with various recombinant viruses produced by these clones; and using these viruses or subunit virus particles for the development of vaccines and the application of diagnostic reagents; and the use of This virus is used as a gene therapy vector or an expression vector.

The advantages of the present invention also include:

The present invention includes various recombinant viruses and subunit virus particle plasmids constructed by molecular biology using these cloned plasmids as mothers.

The present invention also includes various recombinant viruses, subunit virus particles that can be produced using these clones; which contain the cDNAs described above.

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

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

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

The present invention also includes the use of the viral or subviral unit plasmids as gene therapy vectors or expression vector plasmids and viral or subviral particles produced by using these plasmids.

The present invention provides a new tool and approach for detection, prevention and immunization of EV71 virus infection, and provides a possibility for gene therapy and vaccine development by using the EV71 strain infectious clone as a viral vector.

Description of drawings

Figure 1: Construction of an infectious cDNA clone of EV71 strain js1 in which,

(A) Infectious clone construction strategy; Zika virus whole genome schematic diagram, the black bars at both ends represent 5'-NTR and 3'-NTR respectively; the structural protein region and non-structural protein region of the virus are shown in the figure; the full length of the virus The sequence is divided into 4 segments and amplified respectively. The first segment F1 contains the T7 sequence, and the fourth segment F4 contains the polyA ₃₀ sequence introduced by PCR primers; the synthesized sequences are sequentially connected to pACNR by restriction endonucleases as shown in the figure. (B) by fusion PCR, the Nluc or EGFP gene was fused in-frame at the N-terminus of VP4, and an additional amino acid sequence AITTL was added to the C-terminus of the Nluc or EGFP gene to facilitate cleavage by viral protease to produce correct VP4 the N-terminus of .

Figure 2: Infectious cDNA clone of EV71 strain js1 produces virus replication and infectivity, wherein,

Infectious cloning plasmid was used as a template, transcribed into viral RNA in vitro, viral RNA was introduced into Vero cells by electroporation, supernatant virus was collected, titered by plaque assay on Vero cells, and infectious clones were generated (Clone-WT) The comparison between the plaques produced by the parent virus (Parent) and the plaques produced by the parent virus (Parent) is shown in the figure (top), the virus produced by the infectious clone of the same titer and the parent virus were re-infected with Vero cells (MOI=0.1), and the difference after infection was collected. Time (h.p.i) cell supernatant was titrated by plaque assay, and the growth curves of the two were obtained as shown in the figure (bottom), and the virus titer was represented by PFU/ml.

Figure 3: Production of recombinant viruses containing reporter genes Nluc and EGFP, wherein,

(A) Infectious cloned plasmids containing reporter genes Nluc and EGFP, and infectious cloned plasmids without reporter genes, were transcribed into viral RNA in vitro, and the viral RNA was introduced into Vero cells by electroporation, and the supernatant virus was collected, and the virus was collected in Vero cells. Titer titration was carried out using the plaque experiment, and the plaques produced by the recombinant viruses containing each reporter gene were compared with those produced by the virus without the reporter gene; (B) the same titer of the virus containing each reporter gene was different The virus containing the reporter gene was re-infected with Vero cells (MOI=0.1), and the supernatants of different days after infection were collected and titrated by plaque assay to obtain the growth curve; the virus titer was expressed by PFU/ml.

Figure 4: Stability of recombinant virus containing EGFP reporter gene, wherein,

The supernatant of cells infected with recombinant virus EV71-EGFP was diluted 1:10, and then re-infected with new Vero cells. After two days of infection, the cells were observed with a fluorescence microscope and the supernatant was collected, and the new Vero cells were re-infected with a 1:10 dilution. (C+1), two days after infection, the same cells were observed with a fluorescence microscope and the supernatant was collected for re-infection; the infection was successively subcultured, and the expression of EGFP in the infected cells was observed.

Figure 5: Nluc-producing activity of recombinant viruses containing the Nluc reporter gene, wherein,

The infectious cloned plasmid containing the reporter gene Nluc and the plasmid containing the VP1 E145G and 3C C147A mutations were transcribed into viral RNA in vitro, and the viral RNA was introduced into Vero cells by electroporation. The cells were collected at different time points after electroporation, and the intracellular Nluc activity was detected. C147A is a deletion mutation of 3C protease enzymatic activity.

Figure 6: Infectious cDNA clone of EV71 strain js1 produces virus-infected mice to construct an animal infection model, wherein,

(A) Viruses generated from infectious cDNA clones infected different strains of 3-day-old fetal mice (1.4×10 ⁴ pfu/mice) and observed 5 days after infection. (B) Survival curve of mice after virus infection (n=5/group). (C) Survival curves of 3-day-old ICR mice (n=5/group) generated by infectious cDNA clones (WT) and clones carrying the VP1 E145G mutation.

Detailed ways

The methods used in the present invention are all conventional molecular biology methods, and the specific operation details are not repeated here.

Example 1: Construction of an infectious cDNA clone of EV71 strain js1

As shown in Figure 1A, the virus isolated from fecal specimens was cultured in RD cells. When the cells showed obvious cytopathic changes, total cellular RNA was extracted, and the reverse transcriptase was superscript II (Invitrogen) and sequence-specific primers (GCT) were used. AG CGCT ttt tttttttttttt tttttttttt ttttt) was reverse transcribed, and the obtained cDNA was used as a template, and the high-fidelity enzyme super Fi (Invitrogen) was used for PCR amplification in 4 segments, and the amplification primer was 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 tttttttt tttttttt tttttttt ttttttGCT ATTCT GGTTAT AACAA ATTTA CCCCCACCAG), the amplified F4 fragment was first subjected to restriction endonuclease After digestion with NotI/AfeI, it was 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, which was amplified by PCR. The F2 fragment was ligated into pANCR-F34 using PmlI/NruI to obtain pANCR-F234, and the PCR-amplified F1 fragment was ligated into pANCR-F234 using NotI/PmlI to obtain the final full-length cDNA clone, named pEV71-js1.

In order to construct the infectious cloning plasmid with reporter gene EGFP (as shown in Figure 1B), use fusion PCR to fuse three sequences, wherein EGFP-F1 is EV71 5UTR sequence, and PCR amplification primers are S: CCTGA CGTG TCGA CGCGG, SEQ ID NO 18, As: cctc gccct tgctcac CATcatatgG TTTAGCTGT GTTAAG GGTCAAGA, SEQ ID NO 19, EGFP-F2 is a fragment containing EGFP, PCR amplification primer is S: TCTT GACC CTTAAC ACAGC TAA ACcatatgATG gtga gcaag ggcg agg, SEQ ID NO 20, As: CGCT GTGT AGACAC TTGCGA ACCAAGAGTGGTG ATCGC atgcat cttgtac agctcgt ccatgc cg, SEQ ID NO 21, EGFP-F3 is a fragment containing VP4 and VP2 regions, PCR amplification primers are S: cggca tggac gagct gtaca agatgc atGCGA TCACCACT CTTGG TTCGC AAGTG TCTA CACAG CG, SEQ ID NO 22; As: CTGC ACGT GGAT GCA GAACCC, SEQ ID NO 23, after the three fragments were fused by fusion PCR, they were connected into the pEV71-js1 plasmid using NotI/PmlI, and the sequence in the original plasmid was replaced to obtain pEV71 -js1-EGFP plasmid.

In order to construct the infectious cloning plasmid with reporter gene Nluc (as shown in Figure 1B), use fusion PCR to fuse two sequences, wherein Nluc-F1 is the EV715UTR sequence, and the PCR amplification primer is S: CTGC ACGT GGAT GCA GAA CCC , SEQ ID NO 24, As: gaaa tcttcg agtgtga agaccattct agaGTT TAGC TGTG TTA AGGG TCAAG, SEQ ID NO 25, EGFP-F2 is a fragment containing Nluc, and the PCR amplification primer is S: CTTG ACCC TTAAC ACAGCTAA ACtct agaat ggtctt cacac tcgaa gatttc , SEQ ID NO 26; As: CGCat gcatcg ccagaatgcgt tcgca, SEQ ID NO 27. After the two fragments were fused by fusion PCR, they were ligated into the pEV71-js1-EGFP plasmid using NotI/NsiI, and the sequence in the original plasmid was replaced to obtain the pEV71-js1-Nluc plasmid.

Example 2: Infectious cDNA clone of EV71 strain js1 produces virus replication ability and infectivity

The infectious cloning plasmid pEV71-js1 was digested with HindIII, linearized, and then T7 using an in vitro transcription kit (Ambion). In vitro transcribed RNA3g was transferred into Vero cells by electrotransduction. 2 days after electroporation, when the cells became diseased, the virus supernatant was collected, centrifuged at 3000g for 10min, and then filtered through a 0.45m filter to remove cell debris. The virus in the supernatant was titered using a plaque assay. The plaques formed by the virus produced by the infectious cloning plasmid were compared with the plaques of the parent virus originally isolated as shown in Figure 2 (top), and there was no significant difference in the morphology and size of the plaques. The virus produced by the infectious clone of the same titer was re-infected with the parent virus in Vero cells (MOI=0.1), and the cell supernatants at different times after infection were collected and titrated by plaque assay (expressed in PFU/ml), The growth curves of the two are shown in Figure 2 (bottom), and there is no significant difference in the growth curves of the two.

Embodiment 3: the production of the recombinant virus containing reporter gene Nluc and EGFP and its stability

Infectious cloned plasmids containing reporter genes Nluc and EGFP, and infectious cloned plasmids without reporter genes, were transcribed into viral RNA in vitro as above, and electroporated into Vero cells. The virus titer was titrated by plaque assay. As shown in Figure 3A, the plaque morphology and size of the virus containing the reporter genes EGFP and Nluc were similar to the virus without the reporter gene. Vero cells were re-infected (MOI=0.1) with the same titer of the virus containing each reporter gene and the virus without the reporter gene, and the supernatants of different days after infection were collected and titrated by plaque assay. The obtained growth curve , as shown in Figure 3B, compared with the wild virus, the growth cycle of the virus carrying the reporter gene lagged behind, suggesting that the fusion of the reporter gene leads to the delay of the virus replication cycle. The replication ability of the virus produced by the infectious cloning plasmid containing the reporter gene Nluc can be judged by measuring the activity of intracellular Nluc using the Nluc substrate (Promega). The plasmids containing the VP1 E145G and 3C C147A mutations were transcribed into viral RNA in vitro, and the viral RNA was introduced into Vero cells by electroporation, and the intracellular Nluc activity was measured at different times. Viral RNA, after transfection, the activity of intracellular Nluc no longer rises after 8 hours, and its activity only reflects the initial translation signal of viral RNA, but after transfection of wild virus live VP145G viral RNA, Nluc over time appears as Gradually rise, indicating normal virus replication signal. In order to prove the stability of the reporter gene of the virus containing the EGFP reporter gene, we used the supernatant of EV71-EGFP virus-infected cells to be diluted 1:10, and then re-infected new Vero cells, and the cells were observed and collected with a fluorescence microscope two days after infection. The supernatant was re-infected with new Vero cells (C+1) at a dilution of 1:10. After two days of infection, the cells were observed with a fluorescence microscope and the supernatant was collected for re-infection; the infection was successively subcultured, and the expression of EGFP in the infected cells was observed. In some cases, the EGFP gene was still stable after at least 6 consecutive passages.

Example 4: Infectious cDNA cloning of EV71 strain js1 produces virus-infected mice to construct an animal infection model

As shown in Figure 6A, the virus produced by the infectious cDNA clone infected 3-day-old fetal mice of different strains (1.4×10 ⁴ pfu/mice), and the mice were observed 5 days after infection. Compared with the uninfected mice, the mice infected with the virus The mice were all quadriplegic. The survival curves of different strains of mice after infection are shown in Figure 6B, and they all reached 100% mortality within 10 days. The virus produced by the infectious clone carrying the VP1E145G mutation infects 3-day-old ICR mice, which is different from wild-type mice and does not lead to the death of the mice, indicating that the E145 locus is the decisive locus for the lethality of virus-infected mice. Explains the reason why the infection lethality of the passaged virus decreases with the increase of the passage number.

sequence listing

<110> Fudan University

<120> Infectious cDNA Cloning Based on EV71 Strain and Its Application

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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 gggtcattgg aagtcacctt catgtttact 4080

ggatccttca tggctaccgg caagatgctc atagcctata caccgccagg gggtcctctg 4140

cccaaggacc gggcgaccgc catgttgggc acgcacgtca tctgggattt tgggctgcaa 4200

tcgtctgtta cccttgtaat accatggatc agtaacactc attatagagc acatgcccga 4260

gatggagtgt ttgactatta cactacaggg ttagtcagta tatggtacca gacaaattac 4320

gtggttccaa tcggtgcgcc caacacagcc tatataatag cactagcggc agcccaaaag 4380

aacttcacta tgaaattgtg caaggatgct agtgatatcc tgcagacggg caccatccag 4440

ggagataggg tggcagatgt aattgaaagt tccataggag atagcgtgag cagagccctc 4500

actcacgctc taccagcacc cacaggccaa aacacacagg tgagcagtca tcgactggat 4560

acaggcaagg ttccagcact ccaagctgct gaaattgggg catcatcaaa tgctagtgac 4620

gagagcatga ttgaaacacg ttgtgttctt aactcgcata gtacagctga gaccactctt 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 tttatgacgg atatcccaca ttcggagaac acaaacagga gaaagacctt 5100

gaatacgggg catgtcctaa taacatgatg ggtacattct cagtgcggac tgtggggacc 5160

tccaagtcca agtacccttt agtggttagg atttacatga gaatgaagca cgtcagggcg 5220

tggatacctc gcccgatgcg caaccagaac tacctgttca aagccaaccc aaattatgct 5280

ggcaactcta ttaagccaac tggtgccagt cgcacagcga tcaccactct tgggaaattt 5340

ggacaacagt ctggggctat ttatgtgggc 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 ataacttgaa acagctgcca 6240

ctgctagaga atcagatctc gaacttggaa caatctgctg cttcacaaga ggaccttgaa 6300

gtcatgtttg ggaatgtgtc gtacctagct cacttctgtc gcaagtttca accgctatac 6360

gccacggaag ctaaaagagt ctatgccctg gagaagagaa tgaataacta tatgcagttc 6420

aagagcaaac accgaattga acctgtatgt ctcattatta ggggctcacc aggcaccggg 6480

aagtctctag ccactggtat tattgctcga gcaatcgctg ataagtacca ctccagcgtg 6540

tactcgctcc caccagaccc ggatcatttt gacggttaca agcaacaggt ggttacagtg 6600

atggatgatt tgtgtcaaaa ccccgatggt aaggatatgt ccttattctg tcaaatggta 6660

tccaccgtag atttcattcc accaatggct tctctcgagg agaagggagt ttccttcacc 6720

tctaagtttg tcatcgcatc cactaatgcc agtaatatca tagtaccaac agtgtctgat 6780

tctgacgcta ttcgccgcag gttctacatg gactgtgaca ttgaagtgac agactcgtac 6840

aaaacagatc taggtagact ggatgcaggg cgagccgcta aactgtgttc tgaaaataac 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 aatagagcgg 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 cagcactgct 7680

agcgatgcca ccctagtgat caacacggag cacatgccgt caatgtttgt cccggtgggt 7740

gacgttgtgc agtatggctt tttgaatctc agtggcaagc ctacccatcg caccatgatg 7800

tacaattttc ctactaaagc aggacagtgt ggaggagtgg tgacatctgt tgggaaggtt 7860

gtcggtattc acattggtgg caatggcaga caaggttttt gcgcaggcct caaaaggagt 7920

tactttgcta gtgaacaagg agagatccag tgggttaagc ccaataaaga aactggaaga 7980

ctcaacatca atggaccaac ccgcaccaag ttagaaccta gtgtattcca tgacatcttc 8040

gagggaaata aggaaccagc tgtcttgcac agtaaagacc cccgacttga ggtagatttt 8100

gaacaggccc tgttctctaa gtatgtggga aacacactac atgagcctga cgagtacatc 8160

aaagaggcag ctctacatta tgcaaaccaa ttaaagcaac tagaaatcaa tacctctcaa 8220

atgagcatgg aggaggcctg ctatggtact gagaatcttg aggctattga tcttcacact 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

tgaggcatgt ggatacagtg atcgagtggc gcaattaact attggcaact ccaccatcac 1020

gacgcaagaa gcggctaaca tcatagtcgg ctatggtgag tggccttcct actgctcaga 1080

ttctgacgct acagcagtgg ataaaccaac gcgcccggat gtttcagtga acaggtttta 1140

cacattggac actaaattgt gggagaaatc gtccaaggga tggtactgga agttcccgga 1200

tgtgttaact gaaactgggg tttttgggca aaatgcacaa ttccactacc tctaccgatc 1260

agggttctgc atccacgtgc agtgcaatgc cagtaaattc caccaaggag cactcctagt 1320

cgctgtccta ccagagtatg tcattgggac agtggcaggc ggtacaggga cggaagacac 1380

ccacccccccc tacaagcaga cccaacccgg cgccgatggt ttcgagttgc aacacccgta 1440

cgtgcttgat gctggcatcc caatatcaca gttaacagtg tgcccacacc agtggattaa 1500

tttgaggacc aacaattgtg ctacaataat agtgccatac attaacgcac tgccttttga 1560

ttctgccttg aaccattgca actttggcct gttagttgtg cctattagcc cactagacta 1620

cgaccaagga gcaacgccag taatccctat aactatcaca ttggccccaa tgtgctctga 1680

attcgcaggt ctaggcagg cagtcacgca agggttcccc accgagctaa aacctggcac 1740

aaatcaattt ttaaccaccg atgatggcgt ctcagcacct attctaccaa acttccaccc 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 gtttactgga 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

taagcaagtg cttaagaaac ctgctcttcg cacagcaaca gtgcagggtc cgagccttga 5400

ctttgctctc tccctactga gaaggaacat caggcaggtc caaacagacc aagggcattt 5460

caccatgttg ggtgttaggg atcgcttagc agtcctccca cgccactcac aacctggcaa 5520

aaccatttgg attgagcaca aactcgtgaa cgtccttgat gcagttgaac tggtggatga 5580

gcaaggagtc aacctggaat taaccctcat cactcttgac accaacgaga agtttaggga 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 ggttttttgcg caggcctcaa aaggagttac tttgctagtg aacaaggaga 5940

gatccagtgg gttaagccca ataaagaaac tggaagactc aacatcaatg gaccaacccg 6000

caccaagtta gaacctagtg tattccatga catcttcgag 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 agagaacctt 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 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 1200

ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 1260

gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg 1320

tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 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 Gln Val Ser Thr Gln 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 Gln Ser Leu Lys Gln

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 Gln Leu Thr Ile Gly Asn Ser Thr Ile Thr Thr Gln 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 Gln Asn Ala Gln Phe His Tyr Leu Tyr Arg Ser Gly Phe Cys

165 170 175

Ile His Val Gln Cys Asn Ala Ser Lys Phe His Gln Gly Ala Leu Leu

180 185 190

Val Ala Val Leu Pro Glu Tyr Val Ile Gly Thr Val Ala Gly Gly Thr

195 200 205

Gly Thr Glu Asp Thr His Pro Pro Tyr Lys Gln Thr Gln Pro Gly Ala

210 215 220

Asp Gly Phe Glu Leu Gln His Pro Tyr Val Leu Asp Ala Gly Ile Pro

225 230 235 240

Ile Ser Gln Leu Thr Val Cys Pro His Gln Trp Ile Asn Leu Arg Thr

245 250 255

Asn Asn Cys Ala Thr Ile Ile Ile Val Pro Tyr Ile Asn Ala Leu Pro Phe

260 265 270

Asp Ser Ala Leu Asn His Cys Asn Phe Gly Leu Leu Val Val Pro Ile

275 280 285

Ser Pro Leu Asp Tyr Asp Gln Gly Ala Thr Pro Val Ile Pro Ile Thr

290 295 300

Ile Thr Leu Ala Pro Met Cys Ser Glu Phe Ala Gly Leu Arg Gln Ala

305 310 315 320

Val Thr Gln Gly Phe Pro Thr Glu Leu Lys Pro Gly Thr Asn Gln 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 Gln 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 Gln 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 Gln Ser Thr Leu Leu Gly Gln 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 Gln Ser His Leu

945 950 955 960

Met Leu Ala Gln Gly His Ser Glu Pro Gly Asp Cys Gly Gly Ile Leu

965 970 975

Arg Cys Gln 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 Gln 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 Ile Ile Ala Arg Ala Ile 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 Gln

1265 1270 1275 1280

Gln Val Val Thr Val Met Asp Asp Asp Leu Cys Gln Asn Pro Asp Gly Lys

1285 1290 1295

Asp Met Ser Leu Phe Cys Gln Met Val Ser Thr Val Asp Phe Ile Pro

1300 1305 1310

Pro Met Ala Ser Leu Glu Glu Lys Gly Val Ser Phe Thr Ser Lys Phe

1315 1320 1325

Val Ile Ala Ser Thr Asn Ala Ser Asn Ile Ile Val Pro Thr Val Ser

1330 1335 1340

Asp Ser Asp Ala Ile Arg Arg Arg Phe Tyr Met Asp Cys Asp Ile 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 Ile Gln 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 Gln Val Gln Thr

1555 1560 1565

Asp Gln Gly His Phe Thr Met Leu Gly Val Arg Asp Arg Leu Ala Val

1570 1575 1580

Leu Pro Arg His Ser Gln 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 Gln 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 Gln 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 Gln 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 Gln Gly Phe Cys Ala Gly Leu Lys Arg Ser Tyr Phe Ala

1715 1720 1725

Ser Glu Gln Gly Glu Ile Gln Trp Val Lys Pro Asn Lys Glu Thr Gly

1730 1735 1740

Arg Leu Asn Ile Asn Gly Pro Thr Arg Thr Lys Leu Glu Pro Ser Val

1745 1750 1755 1760

Phe His Asp Ile 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 Gln Ala Leu Phe Ser Lys

1780 1785 1790

Tyr Val Gly Asn Thr Leu His Glu Pro Asp Glu Tyr Ile Lys Glu Ala

1795 1800 1805

Ala Leu His Tyr Ala Asn Gln Leu Lys Gln Leu Glu Ile 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

Ile Asp Leu His Thr Ser Ala Gly Tyr Pro Tyr Ser Ala Leu Gly Ile

1845 1850 1855

Lys Lys Arg Asp Ile 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 Ile Asp Lys Ile Lys Lys Gly Lys Ser

1890 1895 1900

Arg Leu Ile 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 Ile

1925 1930 1935

Thr Gly Ser Ala Val Gly Cys Asn Pro Asp Thr Phe Trp Ser Lys Leu

1940 1945 1950

Pro Ile 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 Ile Gly Tyr Ser Glu Glu Ala Ile Ser Leu Ile Glu Gly Ile

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 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 taaactctag aatggtcttc 2760

acactcgaag atttcgttgg ggactggcga cagacagccg gctacaacct ggaccaagtc 2820

cttgaacagg gaggtgtgtc cagtttgttt cagaatctcg gggtgtccgt aactccgatc 2880

caaaggattg tcctgagcgg tgaaaatggg ctgaagatcg acatccatgt catcatcccg 2940

tatgaaggtc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac 3000

cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt aatcgacggg 3060

gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac 3120

ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat cgacgagcgc 3180

ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt gaccggctgg 3240

cggctgtgcg aacgcattct ggcgatgcat gcgatcacca ctcttggttc gcaagtgtct 3300

acacagcgct ccggttctta cgaaaactca aactcagcca ctgagggttc taccataaac 3360

tacaccacca ttaattacta caaagactcc tatgctgcca cagcaggcaa acagagtctc 3420

aagcaggatc cagacaagtt tgcaaatcct gttaaagaca tattcaccga aatggcagcg 3480

ccactgaagt ccccatccgc tgaggcatgt ggatacagtg atcgagtggc gcaattaact 3540

attggcaact ccaccatcac gacgcaagaa gcggctaaca tcatagtcgg ctatggtgag 3600

tggccttcct actgctcaga ttctgacgct acagcagtgg ataaaccaac gcgcccggat 3660

gtttcagtga acaggtttta cacattggac actaaattgt gggagaaatc gtccaaggga 3720

tggtactgga agttcccgga tgtgttaact gaaactgggg tttttgggca aaatgcacaa 3780

ttccactacc tctaccgatc agggttctgc atccacgtgc agtgcaatgc cagtaaattc 3840

caccaaggag cactcctagt cgctgtccta ccagagtatg tcattgggac agtggcaggc 3900

ggtacaggga cggaagacac ccacccccccc tacaagcaga cccaacccgg cgccgatggt 3960

ttcgagttgc aacacccgta cgtgcttgat gctggcatcc caatatcaca gttaacagtg 4020

tgcccacacc agtggattaa tttgaggacc aacaattgtg ctacaataat agtgccatac 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 ggttttttgcg 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 ttagaggtta tacacacctc aaccccacca gaaatctggt 9900

cgtgaatgtg actggtgggg gtaaatttgt tataaccaga atagcaaaaa aaaaaaaaaa 9960

aaaaaaaaaa aaaaaaagct ta 9982

<210> 6

<211> 10187

<212> DNA

<213> Artificial

<400> 6

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 taaaccatat gatggtgagc 2760

aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 2820

aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 2880

accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 2940

accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 3000

ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 3060

gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 3120

atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 3180

tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 3240

gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 3300

cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 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

tatgtcattg ggacagtggc aggcggtaca gggacggaag acacccaccc cccctacaag 4140

cagacccaac ccggcgccga tggtttcgag ttgcaacacc cgtacgtgct tgatgctggc 4200

atcccaatat cacagttaac agtgtgccca caccagtgga ttaatttgag gaccaacaat 4260

tgtgctacaa taatagtgcc atacattaac gcactgcctt ttgattctgc cttgaaccat 4320

tgcaactttg gcctgttagt tgtgcctatt agcccactag actacgacca aggagcaacg 4380

ccagtaatcc ctataactat cacattggcc ccaatgtgct ctgaattcgc aggtcttagg 4440

caggcagtca cgcaagggtt ccccaccgag ctaaaacctg gcacaaatca atttttaacc 4500

accgatgatg gcgtctcagc acctattcta ccaaacttcc accccacccc gtgtatccac 4560

atacctggtg aagttaggaa cttgctagag ttatgccagg tggagaccat tctggaggtt 4620

aacaatgtgc ccacgaatgc cactagctta atggagagac tgcgcttccc ggtctcagca 4680

caagcaggga aaggtgaact gtgtgcggtg tttagagccg atcctgggcg aaatggacca 4740

tggcaatcca ccttactggg ccagttgtgc gggtactaca cccaatggtc agggtcattg 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 actgctagg 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 cggatcattt tgacggttac 7320

aagcaacagg tggttacagt gatggatgat ttgtgtcaaa accccgatgg taaggatg 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 agtgtgtgggg 7680

aaagccatcc aacttagaga tagaaagtct aaagtcagat acagtgtgga tacggtggtt 7740

tcagaactta ttagggaata cagcaatagg tccgccattg gtaacacaat cgaggctctt 7800

ttccaaggtc cacccaagtt caggccaatt aggattagcc ttgaagaaaa accagcccca 7860

gacgctatta gcgatctcct tgctagtgta gatagtgaag aagtgcgcca gtactgcagg 7920

gatcaaggct ggattattcc tgaagctccc accaatgtgg agcggcacct taatagagcg 7980

gtgctcgtca tgcaatccat caccacagta gtggcggttg tttcgttggt gtacgtcatc 8040

tacaagctct ttgcagggtt tcagggtgca tattctggtg ctcctaagca agtgcttaag 8100

aaacctgctc ttcgcacagc aacagtgcag ggtccgagcc ttgactttgc tctctcccta 8160

ctgagaagga acatcaggca ggtccaaaca gaccaagggc atttcaccat gttgggtgtt 8220

agggatcgct tagcagtcct cccacgccac tcacaacctg gcaaaaccat ttggattgag 8280

cacaaactcg tgaacgtcct tgatgcagtt gaactggtgg atgagcaagg agtcaacctg 8340

gaattaaccc tcatcactct tgacaccaac gagaagttta gggatatcac caaattcatc 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

tatggtcttg atcttcccta ctccacttat gtcaaggacg agctacgctc gattgataaa 9180

atcaagaaag ggaagtcccg cctgatcgag gccagtagtc taaatgattc agtgtacctc 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

ggtaaggagt atggtctgac catgacccct gctgataaat ctccttgctt taatgaggtc 9780

aattggggta atgcgacctt cctcaaaagg ggctttttgc ccgatgaaca gtttccattt 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 aaaaaaaaaaa 30

<210> 9

<211> 39

<212> DNA

<213> Artificial

<400> 9

gctagcgctt tttttttttt tttttttttt tttttttttt 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 ttttttttttg 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 acacttgcga accaagagtg gtgatcgcat gcatcttgta cagctcgtcc 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 gtttagctgt gttaagggtc aag 53

<210> 26

<211> 53

<212> DNA

<213> Artificial

<400> 26

cttgaccctt aacacagcta aactctagaa tggtcttcac actcgaagat ttc 53

<210> 27

<211> 27

<212> DNA

<213> Artificial

<400> 27

cgcatgcatc gccagaatgc gttcgca 27

Claims (20)

1. A cDNA, characterized in that it comprises the nucleic acid sequence of the EV71 strain and the nucleic acid sequence of a low copy plasmid backbone;

the nucleic acid sequence of the strain EV71 covers the 5 'to 3' forward polarity sequence of the EV71 virus, including the 5 'and 3' non-coding regions of the virus and one open reading frame encoding viral proteins.

2. The cDNA of claim 1, further comprising a sequence of a reporter gene, luciferase or a fluorescent protein, inserted in the nucleic acid sequence of the EV71 strain.

3. The cDNA according to claim 1, wherein the amino acid sequence of the open reading frame of the viral protein is as shown in SEQ ID NO 4.

4. The cDNA according to claim 1, wherein the coding sequence of the low copy plasmid backbone is as shown in SEQ ID NO 3.

5. The cDNA of claim 1, wherein the EV71 strain has a nucleic acid sequence shown in SEQ ID NO 2.

6. The cDNA according to claim 1, characterized in that its sequence is as shown in SEQ ID NO 1.

7. The expression product of the cDNA according to any one of claims 1 to 6.

8. A recombinant virus comprising a cDNA according to any one of claims 1 to 6.

9. A subgenomic replicon having a cDNA sequence according to any one of claims 1 to 6.

10. A double-stranded DNA capable of producing the cDNA according to any one of claims 1 to 6.

11. A plasmid containing the double-stranded DNA according to claim 10 or a derivative thereof.

12. The plasmid of claim 11, which is capable of transcribing to produce the full-length infectious RNA of the EV71 strain, or a mutant thereof.

13. A vaccine prepared from the plasmid of claim 11 or 12.

14. A viral vector, characterized in that it is prepared according to the plasmid of claim 11 or 12.

15. A viral particle produced from a cDNA clone according to any one of claims 1 to 6 or prepared from a plasmid according to claim 11 or 12.

16. A method for detecting EV71 virus, which comprises using the virus particle according to claim 15.

17. A method for producing an EV71 virus antibody, which comprises using the cDNA according to any one of claims 1 to 6 or the viral particle according to claim 15.

18. The method of claim 17, wherein the viral particle of claim 15 is used to immunize an animal and isolate an antibody, or to screen a human antibody library.

19. A kit for detecting EV71, comprising the cDNA of any one of claims 1 to 6 or the viral particle of claim 15.

20. Use of the cDNA of any one of claims 1 to 6 or the virion construct of claim 15 for the construction of a cell or animal model for further screening for a medicament against viral EV 71.

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