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

Infectious cDNA clone based on EV71 strain and application thereof Download PDF

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CN112094822A
CN112094822A CN201910474088.3A CN201910474088A CN112094822A CN 112094822 A CN112094822 A CN 112094822A CN 201910474088 A CN201910474088 A CN 201910474088A CN 112094822 A CN112094822 A CN 112094822A
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易志刚
宋志刚
张惠英
袁正宏
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Fudan University
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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 clone based on EV71 strain and application thereof
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to construction of infectious cDNA clone based on an EV71 strain (js1) clinically isolated, viruses generated by the cDNA clone and derivative clones thereof, viruses with reporter genes and application of established animal models in research and development of antiviral drugs, research and development of vaccines and virus diagnosis.
Background
The prior art discloses that enteroviruses are a generic term for a class of viruses, including 3 types of Poliovirus (Poliovirus), 23 types of Coxsackie virus (Coxsackie virus a), 6 types of Coxsackie virus (Coxsackie virus B), 31 types of echovirus (ECHO virus), 68-71 types of Enterovirus (Enterovirus), and 67 types in total. The enteroviruses discovered after the traditional typing are named according to the discovery sequence, and the novel enteroviruses discovered now comprise enteroviruses of types 68, 69, 70, 71 and 72. A novel enterovirus type71, abbreviated EV71, belongs to the family picornaviridae, the genus Enterovirus, which was isolated in 1969 from Australia and the United states and in 1973 in Japan and is considered to be the main pathogen of outbreaks of hand-foot-and-mouth disease in children (Schmidt et al.J. Infect Dis 1974,129: 304-309; Hagiwara et al.Intervirology 1978,9: 60-63.). Prior to 1988, the EV71 virus caused an outbreak of hand-foot-and-mouth disease in infants primarily in the United states, Japan, Europe, and Australia (Weng et al microbees insert 2010; 12: 505-10; Tagaya et al Jpn J Med Sci Biol 1975; 28: 231-4; Blumberg 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 J1988; 7: 484-8). Since 1990, the EV71 virus caused a series of outbreaks in Asia-Pacific regions (Chan et al. Clin Infect Dis 2000; 31: 678-83; Tu et al. Emerg Infect Dis 2007; 13: 1733-41; Jeong et al. Arch Virol 2010; 155: 1707-12). By 2014, EV71 infection has spread to various continents and countries worldwide. Studies have shown that EV71 infection is primarily distributed in asian-pacific regions, and there is also a distribution of EV71 infection in north america, south america, europe and australia.
It has been reported that the EV71 virus is a single-stranded positive-strand RNA virus, the genome of which can encode a single long Open Reading Frame (ORF) and which also contains two long noncoding regions, 5'TURs and 3' TURs, in both sections of the genome. 5' TURs contain an Internal Ribosome Entry Site (IRES) that initiates the viral translation process (Hellen et al. genes Dev.2001; 15, 1593-1612). The open reading frame encoded by the virus is processed post-translationally by proteases encoded by the virus itself into individual viral proteins, including the structural proteins VP4, VP2, VP3, VP1 that make up the viral particle and the non-structural proteins 2A,2B,2C,3A,3B,3C and 3D responsible for viral replication (Racaniello, et al.
Studies have reported that primates can be used as infection models for EV 71. Early in 1978, Hashimoto et al reported that 1.8-3.8kg of cynomolgus monkeys, after 9 weeks of isolation, could be infected with a strain of EV71 virus isolated from stool specimens from a3 year old child, with EV71 virus being neurotoxic to the monkey, and showing clinical symptoms of neurological damage on day four of infection, with the degree of damage being positively correlated with viral titer. And EV71 virus can induce the production of serum neutralizing antibodies in monkeys (Hashimoto et al. Arch Virol.1978; 56: 257-61). Zhang et al, using a rhesus monkey of 3-3.5 years old, can establish an animal infection model with symptoms of intracerebral infection, pulmonary edema, hemorrhage with nerve injury, etc., while venous and respiratory infections can directly cause nervous system infections. Thus, models of different research purposes can be obtained by different infection routes (Zhang et al Lab invest.2011; 91: 1337-50). Furthermore, there are rhesus monkey animal models that cause central nervous system diseases (Liu et al. virology.2011; 412: 91-100).
Non-primate animal models of EV71 have also been reported, e.g., mouse-adapted mutant EV71 strain EV71/MP4 can infect ICR mice, developing nerve and lung injury (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), passaged for virus to obtain mice that could be infected with 3 week-sized NOD/SCID mice, adapted to the EV71 strain, and this mouse model was inhibited in natural killer cell function and lacked functional T, B cells. Furthermore, the obtained adapted strain of mouse mainly infects central nervous system, heart and skeletal muscle of animals (Arita et al. J Virol.2008,82(4): 1787-97). Using interferon receptors alpha, beta and gamma deficient immunodeficient mouse AG129 mice, AG129 mice 2 weeks or less old can infect the natural strain of EV71 and exhibit symptoms of acroparalysis before the mice die (Khong et al.j virol.2012,86(4): 2121-31). Three weeks old transgenic mice expressing the EV71 receptor hscabr 2 were successfully infected with the EV71Isehara/Japan/99(Isehara) strain; studies have shown that constructing a mouse model of EV71 requires either a special mouse adapted strain or a genetically deleted or modified mouse.
Studies have also reported that genomic RNA of single positive-strand (positive-strand) RNA viruses is released and can be translated directly as an mRNA template after entering the host cell cytoplasm; the viral nonstructural proteins produced by translation recruit the viral genome to form replication complexes to initiate viral gene replication and life cycle, and therefore the genomic RNA of single positive strand RNA viruses is infectious and, when introduced into a host cell, can completely initiate the entire life cycle of the virus (Racaniello, et al, science.1981,214(4523): 916). Methods for constructing infectious clones generally use total RNA of virus-infected cells as a template, reverse transcribe it to complementary DNA (cDNA), and then clone viral fragments into a cloning vector to form infectious clones of the virus. The constructed infectious clones use in vitro transcription to produce intact viral RNA, which is then transfected into host cells to initiate the viral life cycle, producing progeny virus. Or if the constructed infectious clone has a eukaryotic cell promoter, the infectious clone can be directly transfected with plasmids, and RNA polymerase of host cells transcribes the full-length RNA of the virus, so that the life cycle of the virus is started, and progeny virus is generated.
Mouse model studies demonstrated that glutamate at VP 1145 of EV71is the major site of viral mouse death, and that methylation of lysine at VP 2149 synergistically promoted the ability of VP 1145E to cause mouse death (Huang et al virology.2012,422(1): 132-43). This viral site is susceptible to mutation during in vitro passage of the virus, not 145G, resulting in a reduction in the ability of the virus to infect animals (Yi et al.
Based on the foundation and the current status of the prior art, the inventors of the present application intend to provide infectious cDNA clones based on the EV71 strain and applications thereof.
Disclosure of Invention
The invention aims to provide infectious cDNA clone based on EV71 strain and application thereof based on the foundation and the current situation of the prior art. In particular to a stable infectious cDNA clone of EV71 strain, which can be automatically replicated in cells, generate progeny virus particles and express a reporter gene.
The invention also provides recombinant virus or subunit virus particles, plasmids and the like constructed on the basis of the clone, and provides support for constructing animal models, developing vaccines and developing antiviral drugs.
The EV71 strain (named as js1) is clinically isolated, mouse adaptive mutation is not needed, mice which are not changed in gene background can be infected, virus particles with stable gene sequences can be generated by constructing infectious clone of the strain, common mice are infected, and a simple and efficient EV71 animal infection model is established.
More specifically, the present invention is to provide a novel,
the invention provides a cDNA, which comprises a nucleic acid sequence of EV71 strain and a nucleic acid sequence of a low-copy plasmid skeleton; 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.
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 virus protein open reading frame is shown as SEQ ID NO 4.
The coding sequence of the low-copy plasmid skeleton is shown as SEQ ID NO 3.
The nucleic acid sequence of the EV71 strain is shown as SEQ ID NO 2.
In a preferred embodiment of the invention, the sequence of the infectious cDNA clone of the EV71 strain is shown as SEQ ID NO 1.
In one embodiment of the invention, the construction of a stable infectious cDNA clone of a clinically isolated EV71 strain (nucleic acid sequence 1), derivative clones containing various reporter genes (nucleic acid sequence 5 and nucleic acid sequence 6) and various mutant clones constructed by taking the derivative clones as a parent are provided. The viral RNA produced by these clones is capable of self-replication in cells, producing progeny viral particles, and expressing reporter genes.
The invention also comprises a recombinant virus clone which is constructed by taking the sequence of the nucleic acid sequence 6 or the nucleic acid sequence 7 as a female parent and replacing Nluc or EGFP and contains a heterologous report sequence or a target gene, and a sequence thereof.
The invention also includes various chimeric viruses produced by infectious cloning of various chimeric viruses and cloning of recombinant viruses containing heterologous reporter sequences or target genes and various viral particles containing reporter genes or foreign genes.
The invention also includes the recombinant virus clone which is constructed by the full-length infectious clone sequence and has heterologous resistance sequence inserted into the same open reading frame in the virus protein and the sequence thereof.
Specifically, the invention provides an infectious cDNA clone (nucleic acid sequence 1) containing a clinically isolated EV71 strain (js1), wherein the infectious clone (nucleic acid sequence 1) comprises a nucleic acid sequence (nucleic acid sequence 2) of a full-length EV71 strain (js1) and a low-copy plasmid skeleton (nucleic acid sequence 3). The nucleic acid sequence 2 covers 5 'to 3' positive polarity (positive-sense) sequences of EV71 virus, which comprises 5 'and 3' non-coding regions of the virus and an open reading frame (open reading frame) for coding virus proteins, an open reading frame virus coding protein (protein sequence 4), wherein a reporter gene luciferase NanoLuc (Nluc) and a fluorescent protein EGFP are inserted into the infectious clone (nucleic acid sequence 1) to respectively form an infectious clone with Nluc (nucleic acid sequence 5) and an infectious clone with EGFP (nucleic acid sequence 6), and mutant virus clones (attached viruses), attenuated virus clones (live-attenuated viruses), replication defective virus clones (defective viruses) and replication non-infectious clones (replication-reactive-infectious clones) obtained by means of changing nucleic acids on the basis of the clones, such as a subgenomic replicon comprising a deletion of a structural protein.
The sequences 1 to 6 are specifically as follows:
nucleic acid sequence 1, SEQ ID NO 1:
GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTATAGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACATGGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat
nucleic acid sequence 2, SEQ ID NO 2:
TTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACATGGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGC
nucleic acid sequence 3, SEQ ID NO 3:
AGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGC
protein sequence 4, SEQ ID NO 4:
MGSQVSTQRSGSYENSNSATEGSTINYTTINYYKDSYAATAGKQSLKQDPDKFANPVKDIFTEMAAPLKSPSAEACGYSDRVAQLTIGNSTITTQEAANIIVGYGEWPSYCSDSDATAVDKPTRPDVSVNRFYTLDTKLWEKSSKGWYWKFPDVLTETGVFGQNAQFHYLYRSGFCIHVQCNASKFHQGALLVAVLPEYVIGTVAGGTGTEDTHPPYKQTQPGADGFELQHPYVLDAGIPISQLTVCPHQWINLRTNNCATIIVPYINALPFDSALNHCNFGLLVVPISPLDYDQGATPVIPITITLAPMCSEFAGLRQAVTQGFPTELKPGTNQFLTTDDGVSAPILPNFHPTPCIHIPGEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKGELCAVFRADPGRNGPWQSTLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKMLIAYTPPGGPLPKDRATAMLGTHVIWDFGLQSSVTLVIPWISNTHYRAHARDGVFDYYTTGLVSIWYQTNYVVPIGAPNTAYIIALAAAQKNFTMKLCKDASDILQTGTIQGDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLEGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTLGKFGQQSGAIYVGNFRVVNRHLATHNDWANLVWEDSSRDLLVSSTTAQGCDTIARCDCQTGVYYCNSMRKHYPVSFSKPSLIYVEASEYYPARYQSHLMLAQGHSEPGDCGGILRCQHGVIGIVSTGGNGLVGFADVRDLLWLDEEAMEQGVSDYIKGLGDAFGTGFTDAVSREVEALKNYLIGSEGAVEKILKNLIKLISALVIVIRSDYDMVTLTATLALIGCHGSPWAWIKAKTASILGIPIAQKQSASWLKKFNDMANAAKGLEWVSNKISKFIDWLKEKIVPAAREKVEFLNNLKQLPLLENQISNLEQSAASQEDLEVMFGNVSYLAHFCRKFQPLYATEAKRVYALEKRMNNYMQFKSKHRIEPVCLIIRGSPGTGKSLATGIIARAIADKYHSSVYSLPPDPDHFDGYKQQVVTVMDDLCQNPDGKDMSLFCQMVSTVDFIPPMASLEEKGVSFTSKFVIASTNASNIIVPTVSDSDAIRRRFYMDCDIEVTDSYKTDLGRLDAGRAAKLCSENNTANFKRCSPLVCGKAIQLRDRKSKVRYSVDTVVSELIREYSNRSAIGNTIEALFQGPPKFRPIRISLEEKPAPDAISDLLASVDSEEVRQYCRDQGWIIPEAPTNVERHLNRAVLVMQSITTVVAVVSLVYVIYKLFAGFQGAYSGAPKQVLKKPALRTATVQGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVVGIHIGGNGRQGFCAGLKRSYFASEQGEIQWVKPNKETGRLNINGPTRTKLEPSVFHDIFEGNKEPAVLHSKDPRLEVDFEQALFSKYVGNTLHEPDEYIKEAALHYANQLKQLEINTSQMSMEEACYGTENLEAIDLHTSAGYPYSALGIKKRDILDPTTRDVSRMKFYMDKYGLDLPYSTYVKDELRSIDKIKKGKSRLIEASSLNDSVYLRMAFGHLYEAFHANPGTITGSAVGCNPDTFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYSEEAISLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNIIIRALLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSPCFNEVNWGNATFLKRGFLPDEQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKQEYEKFVSTIRSVPVGRALAIPNYENLRRNWLELF
nucleic acid sequence 5, SEQ ID NO 5:
GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTATAGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACtctagaatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgatgcatGCGATCACCACTCTTGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat
nucleic acid sequence 6, SEQ ID NO 6:
GCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTGTCGACGCGGCCGCTAATACGACTCACTATAGGTTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGTACCTCGGTACCTTTGTGCGCCTGTTTTACACCCCCCCCCCAATGAAACTTAGAAGCAATAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCGTTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTAGCCATCCGGTGTGCAACAGAGCAATTATTTACCTATTTATTGGTTTTGTACCATTAACCTCGAATTCTGTGACCACCCTTAATTATATCTTGACCCTTAACACAGCTAAACcatatgATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagatgcatGCGATCACCACTCTTGGTTCGCAAGTGTCTACACAGCGCTCCGGTTCTTACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAaCAGAGTCTCAAGCAGGATCCAGACAAGTTTGCAAATCCTGTTAAAGACATATTCACcGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCATCACGACGCAAGAAGCGGCTAACATCATAGTCGGCTATGGTGAGTGGCCTTCCTACTGCTCAGATTCTGACGCTACAGCAGTGGATAAACCAACGCGCCCGGATGTTTCAGTGAACAGGTTTTACACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAgCACTcCtAgTCGCTGTCCTACCAGAGTATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGACACCCACCCCCCCTACAAGCAGACCCAACCCGGCGCCGATGGTTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAATTGTGCTACAATAATAGTGCCATACATTAACGCACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGCCTGTTAGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCAACGCCAGTAATCCCTATAACTATCACATTGGCCCCAATGTGCTCTGAATTCGCAGGTCTTAGGCAGGCAGTCACGCAAGGGTTCCCCACCGAGCTAAAACCTGGCACAAATCAATTTTTAACCACCGATGATGGCGTCTCAGCACCTATTCTACCAAACTTCCACCCCACCCCGTGTATCCACATACCTGGTGAAGTTAGGAACTTGCTAGAGTTATGCCAGGTGGAGACCATTCTGGAGGTTAACAATGTGCCCACGAATGCCACTAGCTTAATGGAGAGACTGCGCTTCCCGGTCTCAGCACAAGCAGGGAAAGGTGAACTGTGTGCGGTGTTTAGAGCCGATCCTGGGCGAAATGGACCATGGCAATCCACCTTACTGGGCCAGTTGTGCGGGTACTACACCCAATGGTCAGGGTCATTGGAAGTCACCTTCATGTTTACTGGATCCTTCATGGCTACCGGCAAGATGCTCATAGCCTATACACCGCCAGGGGGTCCTCTGCCCAAGGACCGGGCGACCGCCATGTTGGGCACGCACGTCATCTGGGATTTTGGGCTGCAATCGTCTGTTACCCTTGTAATACCATGGATCAGTAACACTCATTATAGAGCACATGCCCGAGATGGAGTGTTTGACTATTACACTACAGGGTTAGTCAGTATATGGTACCAGACAAATTACGTGGTTCCAATCGGTGCGCCCAACACAGCCTATATAATAGCACTAGCGGCAGCCCAAAAGAACTTCACTATGAAATTGTGCAAGGATGCTAGTGATATCCTGCAGACGGGCACCATCCAGGGAGATAGGGTGGCAGATGTAATTGAAAGTTCCATAGGAGATAGCGTGAGCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGCCAAAACACACAGGTGAGCAGTCATCGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCTGCTGAAATTGGGGCATCATCAAATGCTAGTGACGAGAGCATGATTGAAACACGTTGTGTTCTTAACTCGCATAGTACAGCTGAGACCACTCTTGATAGTTTCTTCAGTAGGGCAGGATTAGTTGGAGAGATAGATCTCCCTCTTGAGGGCACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATATAACAGGTTACGCGCAAATGCGTAGAAAGGTAGAGCTATTCACCTACATGCGTTTTGATGCAGAGTTCACTTTTGTTGCGTGCACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGAGCCCCTAAGCCAGATTCTAGGGAATCCCTTGCATGGCAAACCGCCACCAACCCCTCAGTTTTTGTCAAGCTGTCAGACCCTCCGGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGTGCTTATCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACAGGAGAAAGACCTTGAATACGGGGCATGTCCTAATAACATGATGGGTACATTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGCAACCAGAACTACCTGTTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCCAGTCGCACAGCGATCACCACTCTTGGGAAATTTGGACAACAGTCTGGGGCTATTTATGTGGGCAACTTTAGAGTGGTCAACCGACATCTTGCCACCCATAATGATTGGGCAAATCTTGTTTGGGAAGACAGCTCTCGCGACTTGCTCGTGTCATCCACCACTGCCCAAGGTTGTGACACGATTGCCCGTTGCGATTGCCAGACAGGGGTGTACTACTGTAACTCGATGAGAAAACACTACCCAGTCAGTTTTTCAAAACCCAGCCTGATCTATGTAGAGGCTAGCGAGTATTACCCAGCCAGGTACCAATCACATCTCATGCTCGCACAGGGTCACTCGGAACCTGGTGATTGCGGTGGTATCCTTAGGTGCCAACATGGCGTCATCGGCATAGTGTCTACTGGTGGCAATGGGCTCGTTGGCTTTGCAGACGTCAGAGACCTCTTGTGGTTAGATGAAGAAGCTATGGAACAGGGCGTGTCCGACTACATTAAGGGTCTCGGAGATGCTTTTGGAACAGGCTTCACTGACGCAGTCTCAAGGGAGGTTGAAGCTCTCAAGAACTATCTTATAGGGTCTGAAGGAGCAGTTGAGAAAATTTTGAAAAATCTTATTAAACTAATCTCTGCACTGGTGATTGTGATCAGAAGTGATTACGACATGGTTACCCTCACTGCAACCTTAGCGCTGATAGGTTGTCATGGCAGTCCTTGGGCTTGGATTAAAGCCAAAACAGCCTCCATCTTAGGTATCCCTATCGCCCAAAAGCAGAGCGCTTCCTGGCTCAAGAAGTTCAATGACATGGCCAACGCCGCTAAGGGGTTAGAGTGGGTTTCCAACAAGATCAGCAAATTTATTGATTGGCTTAAGGAGAAAATAGTACCAGCAGCCAGGGAGAAGGTTGAATTCCTAAATAACTTGAAACAGCTGCCACTGCTAGAGAATCAGATCTCGAACTTGGAACAATCTGCTGCTTCACAAGAGGACCTTGAAGTCATGTTTGGGAATGTGTCGTACCTAGCTCACTTCTGTCGCAAGTTTCAACCGCTATACGCCACGGAAGCTAAAAGAGTCTATGCCCTGGAGAAGAGAATGAATAACTATATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTCATTATTAGGGGCTCACCAGGCACCGGGAAGTCTCTAGCCACTGGTATTATTGCTCGAGCAATCGCTGATAAGTACCACTCCAGCGTGTACTCGCTCCCACCAGACCCGGATCATTTTGACGGTTACAAGCAACAGGTGGTTACAGTGATGGATGATTTGTGTCAAAACCCCGATGGTAAGGATATGTCCTTATTCTGTCAAATGGTATCCACCGTAGATTTCATTCCACCAATGGCTTCTCTCGAGGAGAAGGGAGTTTCCTTCACCTCTAAGTTTGTCATCGCATCCACTAATGCCAGTAATATCATAGTACCAACAGTGTCTGATTCTGACGCTATTCGCCGCAGGTTCTACATGGACTGTGACATTGAAGTGACAGACTCGTACAAAACAGATCTAGGTAGACTGGATGCAGGGCGAGCCGCTAAACTGTGTTCTGAAAATAACACTGCAAATTTCAAACGTTGCAGCCCATTAGTGTGTGGGAAAGCCATCCAACTTAGAGATAGAAAGTCTAAAGTCAGATACAGTGTGGATACGGTGGTTTCAGAACTTATTAGGGAATACAGCAATAGGTCCGCCATTGGTAACACAATCGAGGCTCTTTTCCAAGGTCCACCCAAGTTCAGGCCAATTAGGATTAGCCTTGAAGAAAAACCAGCCCCAGACGCTATTAGCGATCTCCTTGCTAGTGTAGATAGTGAAGAAGTGCGCCAGTACTGCAGGGATCAAGGCTGGATTATTCCTGAAGCTCCCACCAATGTGGAGCGGCACCTTAATAGAGCGGTGCTCGTCATGCAATCCATCACCACAGTAGTGGCGGTTGTTTCGTTGGTGTACGTCATCTACAAGCTCTTTGCAGGGTTTCAGGGTGCATATTCTGGTGCTCCTAAGCAAGTGCTTAAGAAACCTGCTCTTCGCACAGCAACAGTGCAGGGTCCGAGCCTTGACTTTGCTCTCTCCCTACTGAGAAGGAACATCAGGCAGGTCCAAACAGACCAAGGGCATTTCACCATGTTGGGTGTTAGGGATCGCTTAGCAGTCCTCCCACGCCACTCACAACCTGGCAAAACCATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAACTGGTGGATGAGCAAGGAGTCAACCTGGAATTAACCCTCATCACTCTTGACACCAACGAGAAGTTTAGGGATATCACCAAATTCATCCCAGAAAATATCAGCACTGCTAGCGATGCCACCCTAGTGATCAACACGGAGCACATGCCGTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTTTTGAATCTCAGTGGCAAGCCTACCCATCGCACCATGATGTACAATTTTCCTACTAAAGCAGGACAGTGTGGAGGAGTGGTGACATCTGTTGGGAAGGTTGTCGGTATTCACATTGGTGGCAATGGCAGACAAGGTTTTTGCGCAGGCCTCAAAAGGAGTTACTTTGCTAGTGAACAAGGAGAGATCCAGTGGGTTAAGCCCAATAAAGAAAcTggAAGACTCAACATCAATGGACCAACCCGCACCAAGTTAGAACCTAGTGTATTCCATGACATCTTCGAGGGAAATAAGGAACCAGCTGTCTTGCACAGTAAAGACCCCCGACTTGAGGTAGATTTTGAACAGGCCCTGTTCTCTAAGTATGTGGGAAACACACTACATGAGCCTGACGAGTACATCAAAGAGGCAGCTCTACATTATGCAAACCAATTAAAGCAACTAGAAATCAATACCTCTCAAATGAGCATGGAGGAGGCCTGCTATGGTACTGAGAATCTTGAGGCTATTGATCTTCACACTAGTGCAGGTTACCCCTATAGTGCCCTAGGGATAAAGAAAAGAGACATCTTAGACCCTACCACCAGGGACGTGAGTAGAATGAAGTTCTACATGGACAAGTATGGTCTTGATCTTCCCTACTCCACTTATGTCAAGGACGAGCTACGCTCGATTGATAAAATCAAGAAAGGGAAGTCCCGCCTGATCGAGGCCAGTAGTCTAAATGATTCAGTGTACCTCAGAATGGCTTTCGGGCATTTGTATGAGGCTTTCCACGCAAATCCTGGGACGATAACTGGATCGGCCGTGGGGTGTAACCCTGACACATTCTGGAGCAAGCTGCCAATTTTGCTCCCTGGTTCACTCTTTGCCTTTGACTACTCAGGCTATGATGCCAGCCTTAGCCCTGTCTGGTTCAGAGCATTAGAATTGGTTCTTAGGGAGATAGGGTATAGTGAAGAGGCAATCTCACTCATTGAGGGAATCAACCACACACATCATGTGTATCGTAATAAGACCTATTGCGTGCTTGGTGGGATGCCCTCAGGCTGTTCAGGAACATCCATCTTCAACTCAATGATCAACAACATTATTATCAGAGCACTGCTCATAAAAACATTTAAGGGCATTGATTTGGATGAACTCAACATGGTCGCTTATGGAGACGATGTGCTCGCTAGCTATCCCTTCCCAATTGATTGCTTGGAACTAGCAAAGACTGGTAAGGAGTATGGTCTGACCATGACCCCTGCTGATAAATCTCCTTGCTTTAATGAGGTCAATTGGGGTAATGCGACCTTCCTCAAAAGGGGCTTTTTGCCCGATGAACAGTTTCCATTTTTGATTCACCCTACTATGCCAATGAGGGAGATCCATGAGTCCATTCGATGGACCAAGGACGCACGGAACACTCAAGATCATGTGCGGTCCTTGTGCCTCCTAGCATGGCATAATGGTAAGCAAGAATACGAGAAGTTTGTGAGCACAATTAGGTCTGTCCCAGTAGGGAGAGCGTTGGCTATTCCAAATTATGAAAATCTTAGACGAAATTGGCTCGAGTTATTTTAGAGGTTATACACACCTCAACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaagcttat。
the present invention also includes expression products of these cDNA clones.
The present invention also includes double-stranded DNA containing the above cDNA, double-stranded DNA (double stranded DNA) capable of generating a full-length infectious clone sequence, positive-sense cDNA (positive-sense cDNA) or negative-sense cDNA (negative-sense cDNA).
The present invention also includes plasmids containing the above-mentioned cDNA or double-stranded DNA.
Preferably, the plasmid can be used for transcribing the full-length infectious RNA of the EV71 strain or a mutant thereof, can be used for generating the plasmid containing the full-length infectious RNA of the full-length EV71 strain (js1) through in vitro transcription, and can be used for generating the plasmid containing the full-length infectious RNA of the full-length EV71 strain (js1) and a derivative plasmid.
Wherein the derivative plasmid comprises:
A. a recombinant virus clone obtained by replacing a partial sequence of the full-length infectious clone of the EV71 strain (js1) of claim 1 with a partial sequence of another isolate (isolates);
B. a mutant virus clone obtained by mutating a sequence in a full-length infection clone of the EV71 strain (js1) according to claim 1 or 2 by using gene mutation;
C. the virus generated by the full-length infection clone of the EV71 strain (js1) is subjected to attenuation (live-attenuated) generated by adaptive mutation, replication non-infection virus (replication reactive non-infections) and non-replication virus (destructive viruses) and other derivative clones.
The present invention provides a plasmid containing the above double-stranded DNA or a derivative thereof.
Preferably, it is capable of transcribing to produce a full-length infectious RNA of the EV71 strain or a mutant thereof.
The present invention also provides a vaccine or viral vector prepared according to the above plasmid;
the present invention provides a viral particle prepared from the above cDNA clone or plasmid;
for example, attenuated (live-infected) virus particles, non-infectious replicating virus (replication competent non-infectious) particles, and non-replicating virus (destructive viruses) particles;
the virus can be separated, purified and subjected to an anti-EV 71 virus antibody by an immune animal method, and can also be used for screening a human antibody library; alternatively, it can be used to prepare the reagent kit for detecting EV71 virus and various cell lines, tissues and animal infection models.
The cell line, tissue and animal infection model can be used for screening the anti-EV 71 virus drugs.
The invention also includes the detection method and preparation method of the virus vector and virus particle;
for example, animals are immunized with the virus particles and antibodies are isolated, or a human antibody library is screened.
In another aspect, the invention provides a kit for detecting EV71, which contains the above-mentioned cDNA or viral particle.
The invention also provides a preparation method of the anti-virus EV71 drug, which uses the cDNA or the virus particles to construct cells or animal models for screening the anti-virus EV71 drug; alternatively, the cDNA or the virus particles are used for constructing cells or animal models for screening the medicine for resisting the virus EV 71.
The infectious clone (nucleic acid sequence 1) of the invention is a complete plasmid (plasmid) consisting of a DNA sequence. The recombinant plasmid contains a nucleic acid sequence (nucleic acid sequence 2) of a full-length EV71 strain (js1) and a low-copy plasmid backbone sequence (nucleic acid sequence 3). Plasmids (plasmids) are closed double-stranded DNA (double stranded DNA) bound by covalent bonds. It contains a sense strand (positive-sense strand) that is identical to the mRNA sequence and a complementary antisense or negative strand (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 a non-translated region (NTR) at the 5' end of the positive strand (positive sense) sequence of the virus, an Open Reading Frame (ORF) and a 3' end untranslated region (3 ' -NTR). In this infectious clone, the 5' end of the full-length viral nucleic acid sequence contains a T7 promoter (TAA TAC GAC TCA CTA TAG G, SEQ ID NO 7) (FIG. 1A), and the full-length viral RNA can be transcribed in vitro by a commercial T7 transcription kit; the 3' end of the full-length viral nucleic acid sequence contained a 30 nucleotide polyA tail (AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA, SEQ ID NO 8) (fig. 1A).
Clinically separating strain to infect RD cells, extracting total RNA of the cells when the cells have cytopathy, and utilizing EV71 specific primer (GCT)AG CGCTtt tttttttt tttttttt ttttttt ttttt, SEQ ID NO 9), and then PCR-amplified using cDNA obtained by reverse transcription (Superscript II reverse transcriptase). Full-length EV71 gene component 4-segment amplification (FIG. 1A), wherein the amplification primer is F1(S: GAC)GC GGCCG CTAA TAC GACTC ACTATAG GTTAAA ACAGC CTGT GGGT TGCAC CC，SEQ ID NO 10；As:GCACTG CACGT GGATGC AGAAC，SEQ ID NO 11)，F2(S:GACGCG GCCGCG TTCT GCAT CCAC GTGCA GTGC，SEQ ID NO 12；As:AAGTC GCGA GAGCT GTCTTC CC，SEQ ID NO 13)，F3(S:GACGCG GCCGCG GGAA GACAG CTCTCG CGACTT，SEQ ID NO 14；As:AATTG TACAT CATG GTGC GATGG GTAGG，SEQ ID NO 15)，F4(S:GACGC GGCCGCCCTAC CCATCG CACCATG ATGTAC AATT，SEQ ID NO 16；As：GCTAGC GCTtttttttt tttttttt tttttttt ttttttGCT ATTCT GGTTAT AACAA ATTTA CCCCCA CCAG, SEQ ID NO 17), cloning the amplified fragment into pANCR vector by using a step-by-step cloning method to obtain a final full-length cDNA clone which is named as pEV71-js1 (FIG. 1A),
this infectious clone was linearized in vitro with HindIII and transcribed to give a polyA tail containing the full length RNA of the virus and its 3' end using the T7 transcription kit. After the virus RNA generated in vitro is introduced into a host cell such as a Vero cell by an electrotransfer or transfection method, the RNA of the virus is used as a translation template to translate ORF of the RNA, and virus polypeptide (protein sequence 4) is generated; the virus polypeptide is processed to form virus structural protein and non-structural protein, and the whole virus life cycle is started to produce progeny virus.
Due to the encoded degeneracy, the same functional protein product can still be obtained by changing the codon without changing the protein sequence; the 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 invention shows strong replication capacity in cells (figure 2), and can be used for infecting cell lines cultured in vitro, nervous tissues, mice (figure 6), monkeys and the like to establish cell models infected by the virus and animal infection models for drug development.
By engineering this infectious clone (nucleic acid sequence 1), a reporter gene is inserted in a specific region of the virus (preceding the VP4 protein coding region), the reporter gene being initiated by translation of the viral IRES. An extra amino acid site (AITTL) was added to the C-terminus of the reporter (FIG. 1B), which is recognized and cleaved by the viral 3C protease, yielding the normal N-terminus of VP 4. The reporter genes, namely, NanoLuc (Nluc) and EGFP (fluorescent protein), were successfully inserted into the infectious clone (nucleic acid sequence 1) to construct an infectious clone with Nluc (nucleic acid sequence 5) and an infectious clone with EGFP (nucleic acid sequence 6), respectively (FIG. 1B). The reporter genes were ligated into pEV71-js1 plasmid (SEQ ID NO: 1) by fusion PCR, designated pEV71-js1-Nluc (SEQ ID NO: 5) (FIG. 1B) and pEV71-js1-EGFP (SEQ ID NO: 6) (FIG. 1C), respectively; the infectious clone was as above, after in vitro HindIII linearization, transcribed viral full-length RNA from T7 transcription kit, and the in vitro transcribed viral RNA was introduced into host cells such as Vero cells by electrotransfer or transfection, and the viral life cycle was initiated to generate progeny virus (FIG. 2). The virus expresses reporter genes Nluc and EGFP in the replication process. Nluc can be detected using a commercial luciferase activity detection kit (fig. 5). The expression of EGFP can be observed with a fluorescence microscope (fig. 4) or detected with a flow cytometer. The resulting progeny virus containing the reporter gene segment re-infects new cells, where it can replicate efficiently. The reporter gene, being in the same open reading frame as the viral protein, is expressed at a level that is responsive to the viral protein level and also to the viral replication level. And the recombinant virus containing the reporter gene continued to pass the reporter gene without loss for a considerable period of time (FIG. 4). The recombinant virus containing the reporter gene can be used for quickly and conveniently detecting the virus replication and packaging level, and can be used for researching the life cycle of the virus, the virus-host interaction, the virus immunology, the development of antiviral drugs and the like.
This infectious clone (nucleic acid sequence 1) is modified so that replication non-infectious virus (replication competent non-infectious virus) such as subgenomic replicon (subgenomic replication) capable of viral gene replication can be constructed by referring to the region of structural proteins VP4-VP3-VP2-VP1 of other viruses of the Enterovirus genus, for example, a knockout virus, but the progeny virus cannot be packaged due to the absence of structural proteins of the virus. Meanwhile, the subgenomic replicon RNA can be trans-complemented (trans-complete) by the expressed structural protein to be packaged into Recombinant Subviral Particles (RSPs) (Barclay, et al.J. Gen Virol.1998,79: 1725-1734; Jia, et al.J. Virol.1998,72:7972-7977), and the subviral particles can be subjected to one-round infection, but can not be packaged again after infection because the genome does not encode the structural protein, so that the subviral particles are non-replicative virus (destructive viruses) particles. These non-replicating viral particles may be used as a form of vaccine.
The infectious clone (nucleic acid sequence 1) is modified to form attenuated virus, and the attenuated virus can be used as vaccine. An attenuated vaccine can be constructed by mutating the 5' NTR with reference to the attenuation strategy of Polio viruses, which are also the family picornaviridae (Arita, et al.J Virol.2008,82: 1787-1797).
The method comprises the steps of infecting a mouse with virus generated by infectious clone, establishing a convenient and stable animal infection model, finding that the lethality of the initially separated strain is reduced after infecting a newborn mouse after more than 3 passages, finding that mutation from E to G occurs at the 145 th position of VP1 of the passaged virus through sequencing, and therefore, the consistency of the mouse model established by infecting the separated virus is poor, and generating the virus by utilizing the infectious clone can ensure that a virus sequence is not influenced by cell passage. The virus obtained by the infectious clone can be used for infecting newborn mice of different strains (ICR, Balb/C, C57) to obtain 100% mortality within 9 days (figure 6B), but the virus carrying the 145G mutation of VP1 can not cause the death of the mice after infecting the mice (figure 6C), and the animal model can be conveniently used for antiviral drug vaccine evaluation and the like.
Novel Enterovirus 71 (Human enterovirus type71, EV71) belongs to the family Microviridae in the family Microviridae (picornaviridae) Is a member of the enterovirus group (enterovirus) of (1). EV71is the major causative agent of hand-foot-and-mouth disease in children worldwide, which can cause children to suffer from mild and severe hand-foot-and-mouth disease. The virus can infect and cause damage to the central nervous system, but the mechanism is unknown. There is currently no effective antiviral drug for the treatment of EV 71. The invention constructs the full-length cDNA clone of the stable virus by separating a clinical EV71 strain and utilizing molecular cloning, and the virus RNA from the cDNA clone can generate the EV71 virus through in vitro transcription RNA and transfection of Vero cells; further, the recombinant virus containing the reporter genes Gluc (Gaussia luciferase) and EGFP is constructed, and the recombinant virus containing the reporter genes Gluc and EGFP is proved to have the capacity of infecting host cells and causing cytopathic effect,the EV71 virus from infectious clone is used to infect the immune-competent ICR, Bab/C and C57 suckling mice, and 100 percent of infected mice die due to nerve injury symptoms within 10 days.
The invention 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 has the following advantages:
the present invention includes various recombinant virus and subunit virus particle plasmids constructed through molecular biology with these cloned plasmids as female parent.
The invention also includes various recombinant viruses, subunit viral particles that can be produced using these clones; which contains the above cDNA.
The invention also includes animal infection models constructed using various recombinant viruses that these clones can produce.
The invention also includes the use of these viral or subunit viral particles and animal models for vaccine development and diagnostic reagents.
The invention also includes the use of these viruses or subunit viral particles to establish animal models for vaccine development and antiviral drug development.
The invention also includes the use of the viral or subviral unit plasmids as gene therapy vectors or expression vector plasmids and the viral or subviral particles produced using these plasmids.
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.
Drawings
FIG. 1 construction of an infectious cDNA clone of EV71 strain js1, in which,
(A) infectious clone construction strategy; zika virus complete genome pattern diagram, two ends of the black column respectively represent 5 '-NTR and 3' -NTR; the virus structural protein region and the non-structural protein region are shown in the figure; the full-length viral sequence is divided into 4 segments for amplification, wherein the first segment F1 contains a T7 sequence, and the fourth segment F4 contains polyA introduced by PCR primers30A sequence; the synthesized sequence is sequentially connected and connected into a pACNR vector through restriction endonucleases according to the figure to obtain full-length clone; (B) by fusion PCR, Nluc or EGFP gene is fused in-frame at the N-terminal of VP4, and an additional amino acid sequence AITTL is added at the C-terminal of the Nluc or EGFP gene, so that the N-terminal of VP4 can be conveniently cut by viral protease.
FIG. 2 infectious cDNA clone of EV71 strain js1 shows the replication and infection capabilities of the virus,
infectious Clone plasmid as template, through in vitro transcription into virus RNA, viral RNA through electrotransfer into Vero cell, collect supernatant virus, in Vero cell using plaque experiment to perform titer titration, infectious Clone produced (Clone-WT) plaque and Parent virus (Parent) produced by the comparison of the plaque as shown in figure (upper), infectious Clone produced virus and Parent virus again infected Vero cell (MOI 0.1), after infection, collecting different time (h.p.i) cell supernatant, using plaque experiment to titrate, get the two growth curves as shown in figure (lower), virus titer is expressed by PFU/ml.
FIG. 3 production of recombinant virus containing reporter genes Nluc and EGFP, wherein,
(A) the infectious clone plasmid containing the reporter genes Nluc and EGFP is transcribed into virus RNA in vitro with the infectious clone plasmid without the reporter genes, the virus RNA is introduced into Vero cells through electrotransfer, supernatant viruses are collected, titer titration is carried out on the Vero cells by utilizing a plaque experiment, and plaques generated by recombinant viruses containing the reporter genes are compared with plaques generated by viruses without the reporter genes; (B) infecting Vero cells again with the same titer of virus containing each reporter gene and the virus without the reporter gene (MOI ═ 0.1), collecting supernatants at different days after infection, titrating the supernatants by using a plaque experiment to obtain a growth curve; viral titers were expressed as PFU/ml.
FIG. 4 stability of recombinant viruses containing EGFP reporter gene, wherein,
diluting cell supernatant infected by the recombinant virus EV71-EGFP at a ratio of 1:10, re-infecting new Vero cells, observing the cells with a fluorescence microscope after two days of infection, collecting supernatant, re-infecting new Vero cells (C +1) at a ratio of 1:10, observing the cells with the fluorescence microscope after two days of infection, collecting supernatant, and re-infecting the cells; and (5) sequentially carrying out passage infection, and observing the expression condition of the EGFP in the infected cells.
FIG. 5 Nluc-producing Activity of recombinant viruses containing Nluc reporter gene, wherein,
the infectious clone plasmid containing the reporter gene Nluc and the plasmid containing VP 1E 145G and 3C C147A mutations are transcribed into virus RNA in vitro, the virus RNA is introduced into Vero cells through electrotransfer, the cells are collected at different time points after the electrotransfer, the activity of Nluc in the cells is detected, and C147A is 3C protease activity deletion mutation.
FIG. 6 infectious cDNA clone of EV71 strain js1 producing virus-infected mice an animal infection model was constructed in which,
(A) virus generated by infectious cDNA clones infected different strains of 3-day-old fetal mice (1.4X 10)4pfu/mouse), observed 5 days after infection. (B) Survival curves of mice after viral infection (n ═ 5/group). (C) Infectious cDNA clone (WT) and clone carrying the VP 1E 145G mutation gave a virus that infected 3-day-old ICR mice with a survival curve (n 5/group).
Detailed Description
The methods used in the present invention are all conventional molecular biology methods, and the details of the operation are not repeated.
Example 1 construction of an infectious cDNA clone of EV71 strain js1
As shown in FIG. 1A, the virus isolated from the stool specimen was cultured in RD cells, and when the cells showed significant cytopathic effects, total cellular RNA was extracted and reverse transcribed using superscript II (Invitrogen)Enzyme, with sequence specific primer (GCT)AG CGCTttt tttttttttttt tttttttttt ttttt) and performing reverse transcription, using the obtained cDNA as a template, performing PCR amplification in 4 sections by using high fidelity enzyme super Fi (Invitrogen), wherein the amplification primer is F1(S: GAC)GC GGCCG CTAA TAC GACTC ACTATAG GTTAAA ACAGC CTGT GGGT TGCAC CC；As:GCACTG CACGT GGATGC AGAAC)，F2(S:GACGCG GCCGCG TTCT GCAT CCAC GTGCA GTGC；As:AAGTC GCGA GAGCT GTCTTC CC)，F3(S:GACGCG GCCGCG GGAA GACAG CTCTCG CGACTT；As:AATTG TACAT CATG GTGC GATGG GTAGG)，F4(S:GACGC GGCCGCCCTAC CCATCG CACCATG ATGTAC AATT；As：GCTAGC GCTtttttttt tttttttt tttttttt ttttttGCT ATTCT GGTTAT AACAA ATTTA CCCCCA CCAG), the amplified F4 fragment was digested with restriction enzymes NotI/AfeI, and ligated to pANCR vector digested with the same restriction enzymes to obtain pANCR-F4 plasmid, the PCR amplified F3 fragment was ligated to pANCR-F4 with NruI/BsrGI to obtain pANCR-F34 plasmid, the PCR amplified F2 fragment was ligated to pANCR-F34 with PmlI/NruI to obtain pANCR-F234, and the PCR amplified F1 fragment was ligated to pANCR-F234 with NotI/PmlI to obtain the final full-length cDNA clone designated as pEV71-js 1.
In order to construct an infectious clone plasmid with a reporter gene EGFP (As shown in FIG. 1B), three sequences are fused by utilizing fusion PCR, wherein EGFP-F1 is an EV715UTR sequence, 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 segment containing EGFP, and the PCR amplification primers are S: TCTT GACC CTTAAC ACAGC TAA ACcata tgATG gtga gcaag ggcg agg, SEQ ID NO 20, As: CGCT GTGT AGACAC TTGCGA ACCAAG AGTGGTG 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 TCAC CACT CTTGG TTCGC AAGTG TCTA CACAG CG, SEQ ID NO 22; CTGC ACGT GGAT GCA GAA CCC, SEQ ID NO 23, and after the three fragments are fused by fusion PCR, the NotI/PmlI is used for connecting into pEV71-js1 plasmid to replace the sequence in the original plasmid, thus obtaining pEV71-js1-EGFP plasmid.
In order to construct an infectious clone plasmid (shown in figure 1B) with a reporter gene Nluc, fusion PCR is utilized to fuse two sequences, wherein Nluc-F1 is an EV715UTR sequence, PCR amplification primers are S: CTGC ACGT GGAT GCA GAA CCC, SEQ ID NO 24, As: gaaa tcttcg agtgtga agaccattct agaGTT TAGC TGTG TTA AGGG TCA AG, SEQ ID NO 25, EGFP-F2 is a fragment containing Nluc, and PCR amplification primers are S: CTTG ACCC TTAAC ACAG CTAA ACtct agaat ggtctt cacac tcgaa gatttc, SEQ ID NO 26; as CGCat gcatcg ccaga atgcgt tcgca, SEQ ID NO 27. After the two fragments are fused by fusion PCR, NotI/NsiI is used for connecting into pEV71-js1-EGFP plasmid, and the sequence in the original plasmid is replaced, so that pEV71-js1-Nluc plasmid is obtained.
Example 2 infectious cDNA clone of EV71 strain js1 produces viral replication and infection
The infectious cloning plasmid pEV71-js1 was digested with HindIII, linearized, and then T7 using an in vitro transcription kit (Ambion). The in vitro transcribed RNA3g was transferred into Vero cells by the method of electrical transduction. After 2 days of electrotransfer, when the cells are diseased, the virus supernatant is collected, centrifuged at 3000g for 10min, and then filtered through a 0.45m filter membrane to remove cell debris. The virus in the supernatant was titrated using a plaque assay. The plaques formed by the virus produced by the infectious cloned plasmid were compared with the plaques of the original isolated parent virus as shown in FIG. 2 (top), and there was no significant difference in the morphology and size of the plaques. The Vero cells were re-infected with the same titer of virus produced by the infectious clone as the parent virus (MOI ═ 0.1), and cell supernatants at different times after infection were collected and titrated by plaque assay (expressed as PFU/ml), and the growth curves of both were shown in fig. 2 (bottom), and there was no significant difference between the growth curves.
Example 3 production of recombinant viruses containing reporter genes Nluc and EGFP and their stability
The infectious clone plasmid containing the reporter genes Nluc and EGFP and the infectious clone plasmid without the reporter genes are transcribed into virus RNA in vitro in the same way, are electrically transferred into Vero cells, the virus in the cell supernatant is collected after two days, and the virus titer is titrated on the Vero cells by using a plaque experiment. As shown in FIG. 3A, the plaques of the viruses containing the reporter genes EGFP and Nluc are similar in morphology and size to those of the viruses not containing the reporter genes. Vero cells (MOI 0.1) were re-infected with the same titer of viruses containing the reporter genes and viruses not containing the reporter genes, supernatants were collected at different days after infection, and titrated by plaque assay to obtain growth curves, as shown in FIG. 3B, in which the growth cycle of the viruses carrying the reporter genes was shown to be delayed compared to the wild viruses, suggesting that fusion of the reporter genes results in a delay in the replication cycle of the viruses. The replication ability of the virus produced by the infectious clone plasmid containing the reporter gene Nluc can be judged by measuring the intracellular Nluc activity using the Nluc substrate (Promega). The plasmid containing mutations of VP 1E 145G and 3C C147A was transcribed in vitro to viral RNA, which was then introduced into Vero cells by electroporation and the intracellular Nluc activity was measured at different times, as shown in fig. 5, viral RNA containing a 3C protease inactivation mutation (C147A) showed no increase in intracellular Nluc activity after 8 hours after transfection and only responded to the initial translation signal of viral RNA, whereas Nluc appeared to gradually increase with time after transfection of wild-type viral live VP145G viral RNA, indicating a normal viral replication signal. To demonstrate the stability of reporter genes of viruses containing EGFP reporter genes, cell supernatants infected by EV71-EGFP virus are diluted by 1:10, new Vero cells are re-infected, the cells are observed by a fluorescence microscope after two days of infection and the supernatants are collected, the new Vero cells (C +1) are re-infected by 1:10 dilution, and the cells are observed by the fluorescence microscope and the supernatants are collected for re-infection after two days of infection; after successive passages, the expression of EGFP in the infected cells is observed, and the EGFP gene is still stable after at least 6 passages.
Example 4 cloning of infectious cDNA of EV71 strain js1 Generation of Virus-infected mice to construct animal infection model
As shown in FIG. 6A, the virus produced by the infectious cDNA clone infected different strains of 3-day-old fetal mice (1.4X 10)4pfu/mouse), mice were observed 5 days after infection, and mice infected with virus all appeared quadriplegia compared to uninfected mice. Survival curves of mice of different strains after infection are shown in fig. 6B, and within 10 days, 100% mortality is achieved. Portable bagThe virus generated by the infectious clone with the VP 1E 145G mutation does not cause death of mice after infecting ICR mice of 3 days old, which indicates that the E145 site is a decisive site for death of virus infected mice and explains the reason that the death rate of infection of the passaged virus is reduced along with the increase of the passage number.
Sequence listing
<110> university of Compound Dan
<120> infectious cDNA clone based on EV71 strain and application thereof
<130> 20190601
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9446
<212> DNA
<213> Artificial
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gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct 60
tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata 120
tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa 180
atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa 240
gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg 300
aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt 360
ttcccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat 420
tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 480
ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 540
ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 600
ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 660
gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 720
gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 780
gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaaggg gtctgacgct 840
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 900
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 960
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 1020
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 1080
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 1140
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 1200
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 1260
aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 1320
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 1380
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 1440
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 1500
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 1560
cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 1620
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 1680
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 1740
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 1800
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 1860
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 1920
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt gtcgacgcgg 1980
ccgctaatac gactcactat aggttaaaac agcctgtggg ttgcacccac tcacagggcc 2040
tactgggcgc aagcactctg gtacctcggt acctttgtgc gcctgtttta cacccccccc 2100
ccaatgaaac ttagaagcaa taaaccacga tcaatagcag gcataacgct ccagttatgt 2160
cttgatcaag cacttctgtt tccccggact gagtatcaat agactgctcg cgcggttgaa 2220
ggagaaaacg ttcgttatcc ggctaactac ttcggaaaac ctagtaacac catgaaagtt 2280
gcggagagct tcgttcagca ctcccccagt gtagatcagg tcgatgagtc accgcgttcc 2340
ccacgggcga ccgtggcggt ggctgcgttg gcggcctgcc catggggtaa cccatggggc 2400
gctctaatac ggacatggtg tgaagagtct actgagctag ttggtagtcc tccggcccct 2460
gaatgcggct aatcccaact gcggagcaca cgcccacaag ccagcgggta gtgtgtcgta 2520
acgggtaact ctgcagcgga accgactact ttgggtgtcc gtgtttcctt ttatctttat 2580
attggctgct tatggtgaca attaaagaat tgttaccata tagctattgg attagccatc 2640
cggtgtgcaa cagagcaatt atttacctat ttattggttt tgtaccatta acctcgaatt 2700
ctgtgaccac ccttaattat atcttgaccc ttaacacagc taaacatggg ttcgcaagtg 2760
tctacacagc gctccggttc ttacgaaaac tcaaactcag ccactgaggg ttctaccata 2820
aactacacca ccattaatta ctacaaagac tcctatgctg ccacagcagg caaacagagt 2880
ctcaagcagg atccagacaa gtttgcaaat cctgttaaag acatattcac cgaaatggca 2940
gcgccactga agtccccatc cgctgaggca tgtggataca gtgatcgagt ggcgcaatta 3000
actattggca actccaccat cacgacgcaa gaagcggcta acatcatagt cggctatggt 3060
gagtggcctt cctactgctc agattctgac gctacagcag tggataaacc aacgcgcccg 3120
gatgtttcag tgaacaggtt ttacacattg gacactaaat tgtgggagaa atcgtccaag 3180
ggatggtact ggaagttccc ggatgtgtta actgaaactg gggtttttgg gcaaaatgca 3240
caattccact acctctaccg atcagggttc tgcatccacg tgcagtgcaa tgccagtaaa 3300
ttccaccaag gagcactcct agtcgctgtc ctaccagagt atgtcattgg gacagtggca 3360
ggcggtacag ggacggaaga cacccacccc ccctacaagc agacccaacc cggcgccgat 3420
ggtttcgagt tgcaacaccc gtacgtgctt gatgctggca tcccaatatc acagttaaca 3480
gtgtgcccac accagtggat taatttgagg accaacaatt gtgctacaat aatagtgcca 3540
tacattaacg cactgccttt tgattctgcc ttgaaccatt gcaactttgg cctgttagtt 3600
gtgcctatta gcccactaga ctacgaccaa ggagcaacgc cagtaatccc tataactatc 3660
acattggccc caatgtgctc tgaattcgca ggtcttaggc aggcagtcac gcaagggttc 3720
cccaccgagc taaaacctgg cacaaatcaa tttttaacca ccgatgatgg cgtctcagca 3780
cctattctac caaacttcca ccccaccccg tgtatccaca tacctggtga agttaggaac 3840
ttgctagagt tatgccaggt ggagaccatt ctggaggtta acaatgtgcc cacgaatgcc 3900
actagcttaa tggagagact gcgcttcccg gtctcagcac aagcagggaa aggtgaactg 3960
tgtgcggtgt ttagagccga tcctgggcga aatggaccat ggcaatccac cttactgggc 4020
cagttgtgcg ggtactacac ccaatggtca 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
ccaccccccc 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 cttaggcagg 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 ggtttttgcg 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 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 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 ccaccccccc 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 ggtttttgcg caggcctcaa aaggagttac 8460
tttgctagtg aacaaggaga gatccagtgg gttaagccca ataaagaaac tggaagactc 8520
aacatcaatg gaccaacccg caccaagtta gaacctagtg tattccatga catcttcgag 8580
ggaaataagg aaccagctgt cttgcacagt aaagaccccc gacttgaggt agattttgaa 8640
caggccctgt tctctaagta tgtgggaaac acactacatg agcctgacga gtacatcaaa 8700
gaggcagctc tacattatgc aaaccaatta aagcaactag aaatcaatac ctctcaaatg 8760
agcatggagg aggcctgcta tggtactgag aatcttgagg ctattgatct tcacactagt 8820
gcaggttacc cctatagtgc cctagggata aagaaaagag acatcttaga ccctaccacc 8880
agggacgtga gtagaatgaa gttctacatg gacaagtatg gtcttgatct tccctactcc 8940
acttatgtca aggacgagct acgctcgatt gataaaatca agaaagggaa gtcccgcctg 9000
atcgaggcca gtagtctaaa tgattcagtg tacctcagaa tggctttcgg gcatttgtat 9060
gaggctttcc acgcaaatcc tgggacgata actggatcgg ccgtggggtg taaccctgac 9120
acattctgga gcaagctgcc aattttgctc cctggttcac tctttgcctt tgactactca 9180
ggctatgatg ccagccttag ccctgtctgg ttcagagcat tagaattggt tcttagggag 9240
atagggtata gtgaagaggc aatctcactc attgagggaa tcaaccacac acatcatgtg 9300
tatcgtaata agacctattg cgtgcttggt gggatgccct caggctgttc aggaacatcc 9360
atcttcaact caatgatcaa caacattatt atcagagcac tgctcataaa aacatttaag 9420
ggcattgatt tggatgaact caacatggtc gcttatggag acgatgtgct cgctagctat 9480
cccttcccaa ttgattgctt ggaactagca aagactggta aggagtatgg tctgaccatg 9540
acccctgctg ataaatctcc ttgctttaat gaggtcaatt ggggtaatgc gaccttcctc 9600
aaaaggggct ttttgcccga tgaacagttt ccatttttga ttcaccctac tatgccaatg 9660
agggagatcc atgagtccat tcgatggacc aaggacgcac ggaacactca agatcatgtg 9720
cggtccttgt gcctcctagc atggcataat ggtaagcaag aatacgagaa gtttgtgagc 9780
acaattaggt ctgtcccagt agggagagcg ttggctattc caaattatga aaatcttaga 9840
cgaaattggc tcgagttatt 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 actgctagag aatcagatct cgaacttgga acaatctgct 7020
gcttcacaag aggaccttga agtcatgttt gggaatgtgt cgtacctagc tcacttctgt 7080
cgcaagtttc aaccgctata cgccacggaa gctaaaagag tctatgccct ggagaagaga 7140
atgaataact atatgcagtt caagagcaaa caccgaattg aacctgtatg tctcattatt 7200
aggggctcac caggcaccgg gaagtctcta gccactggta ttattgctcg agcaatcgct 7260
gataagtacc actccagcgt gtactcgctc ccaccagacc cggatcattt tgacggttac 7320
aagcaacagg tggttacagt gatggatgat ttgtgtcaaa accccgatgg taaggatatg 7380
tccttattct gtcaaatggt atccaccgta gatttcattc caccaatggc ttctctcgag 7440
gagaagggag tttccttcac ctctaagttt gtcatcgcat ccactaatgc cagtaatatc 7500
atagtaccaa cagtgtctga ttctgacgct attcgccgca ggttctacat ggactgtgac 7560
attgaagtga cagactcgta caaaacagat ctaggtagac tggatgcagg gcgagccgct 7620
aaactgtgtt ctgaaaataa cactgcaaat ttcaaacgtt gcagcccatt agtgtgtggg 7680
aaagccatcc aacttagaga tagaaagtct aaagtcagat acagtgtgga tacggtggtt 7740
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 aaaaaaaaaa 30
<210> 9
<211> 39
<212> DNA
<213> Artificial
<400> 9
gctagcgctt tttttttttt tttttttttt ttttttttt 39
<210> 10
<211> 55
<212> DNA
<213> Artificial
<400> 10
gacgcggccg ctaatacgac tcactatagg ttaaaacagc ctgtgggttg caccc 55
<210> 11
<211> 22
<212> DNA
<213> Artificial
<400> 11
gcactgcacg tggatgcaga ac 22
<210> 12
<211> 33
<212> DNA
<213> Artificial
<400> 12
gacgcggccg cgttctgcat ccacgtgcag tgc 33
<210> 13
<211> 22
<212> DNA
<213> Artificial
<400> 13
aagtcgcgag agctgtcttc cc 22
<210> 14
<211> 33
<212> DNA
<213> Artificial
<400> 14
gacgcggccg cgggaagaca gctctcgcga ctt 33
<210> 15
<211> 28
<212> DNA
<213> Artificial
<400> 15
aattgtacat catggtgcga tgggtagg 28
<210> 16
<211> 39
<212> DNA
<213> Artificial
<400> 16
gacgcggccg ccctacccat cgcaccatga tgtacaatt 39
<210> 17
<211> 73
<212> DNA
<213> Artificial
<400> 17
gctagcgctt tttttttttt tttttttttt tttttttttg ctattctggt tataacaaat 60
ttacccccac cag 73
<210> 18
<211> 18
<212> DNA
<213> Artificial
<400> 18
cctgacgtgt cgacgcgg 18
<210> 19
<211> 49
<212> DNA
<213> Artificial
<400> 19
cctcgccctt gctcaccatc atatggttta gctgtgttaa gggtcaaga 49
<210> 20
<211> 49
<212> DNA
<213> Artificial
<400> 20
tcttgaccct taacacagct aaaccatatg atggtgagca agggcgagg 49
<210> 21
<211> 66
<212> DNA
<213> Artificial
<400> 21
cgctgtgtag 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.
CN201910474088.3A 2019-06-02 2019-06-02 Infectious cDNA clone based on EV71 strain and application thereof Pending CN112094822A (en)

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