CN109207491A - A kind of M1 virus full length infection clones and preparation method and its preparing the application in M1 virus - Google Patents
A kind of M1 virus full length infection clones and preparation method and its preparing the application in M1 virus Download PDFInfo
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Abstract
The invention discloses a kind of M1 virus full length infection clones and preparation method and its preparing the application in M1 virus.The M1 virus full length infection clones, sequence are shown in SEQ ID NO.6.Full-length infectious clone successfully prepares M1 virus.The present invention successfully obtains the full-length infectious clone of reverse genetics system-that can be used for preparing the various M1 viruses for recombinating M1 viruses, is with a wide range of applications in terms of neural circuitry label, tumorigenic infection and elimination, the foundation of medicine sorting platform, the analysis of Drug inhibition virus function mechanism, the research and development of vaccine and diagnostic reagent, the foundation of animal model, virus replication and pathogenic mechanism.
Description
Technical field
The invention belongs to field of biotechnology, be more particularly to a kind of M1 virus full length infection clones and preparation method and
It is preparing the application in M1 virus.
Background technique
M1 virus belongs to Togaviridae alphavirus, and genome is single-stranded positive RNA, and length is about 12kb.Gene
The polyprotein of group coding can be cut into 5 structural proteins (C, E3, E2,6K and E1) and 4 non-structural proteins (nsP1,
NsP2, nsP3 and nsP4).Although virus discovery has had more long-range history, Coupling effects, disease with cell
The replicanism of poison etc. is unclear.The reverse genetics system of virus includes: full-length infectious clone, replicon and virus-like
Particle etc..These systems are for fully understanding the Coupling effects of virus and host, the replicanism of virus, and transformation virus tool
It is of great significance.However so far, it there is no the reverse genetics system of M1 virus, to limit relevant grind
Study carefully.
With the continuous development of molecular biology, scientist has been able to the reversed something lost that virus is established by serial of methods
System is passed, and further carrys out directional transformation virus, provides good tool to carry out relevant virological investigation in a deep going way.Cause
This, the present invention is respectively adopted different technological approaches and obtains M1 virus full length infection clones.It will be marked in neural circuitry, sense
Dye and tumors destroyed cell, the foundation of medicine sorting platform, analysis, viral vaccine and the diagnosis of Drug inhibition virus function mechanism
The research and development of reagent, the foundation of animal model and virus replication and the analysis of pathogenic mechanism etc. be with a wide range of applications and
Prospect.
Summary of the invention
The object of the present invention is to provide a kind of full-length infectious clone of M1 virus, sequence is SEQ ID NO:6
It is shown.
The object of the present invention is to provide a kind of preparation methods of M1 virus full length infection clones, and method is simple, easily
Row.
The object of the present invention is to provide a kind of M1 virus full length infection clones applications, which can
Be used to prepare M1 virus, the M1 virus of preparation can be applied to brain science research, infected tumor's cell and kill, drug screening and resist
Former epitope analysis, also in the research and development of the analysis of Drug inhibition virus function mechanism, vaccine and diagnostic reagent, the foundation of animal model
It is with a wide range of applications with virus replication and the analysis of pathogenic mechanism etc..
In order to achieve the goal above, the present invention adopts the following technical scheme:
The building of M1 virus full length infection clones:
The method of digestion and homologous recombination is respectively adopted by four segment F1 of M1 viral genome (SEQ ID NO:1), F2
(SEQ ID NO:2), F3 (SEQ ID NO:3) and F4 (SEQ ID NO:4) are sequentially inserted into carrier F5's (SEQ ID NO:5)
Between PacI and RsrII restriction enzyme site, the correct clone designation of the sequencing of acquisition is M1-F1-F2-F3-F4, as M1 virus
Full-length infectious clone pFLM1, sequence are shown in SEQ ID NO.6.
The preparation of M1 virus:
1. full-length infectious clone pFLM1 is transfected BHK21 cell culture, viral supernatants are collected in different time；
2. using MluI digestion pFLM1, the product being digested is recycled, MEGAscript T7Transcription Kit is used
PFLM1 after digestion is transcribed in vitro becomes RNA, and transfects BHK21 cell, and 37 DEG C, 5% (v/v) CO2It is cultivated in incubator, and
The cell that untransfected RNA is arranged simultaneously as a control group, passes through the cell state of microscope observation experiment group and control group.
M1 virus made from above method can be applied to brain science research, infected tumor's cell and kill, drug screening and
Characterization of antigenic epitopes is also built in the research and development of the analysis of Drug inhibition virus function mechanism, vaccine and diagnostic reagent, animal model
Vertical and virus replication and the analysis of pathogenic mechanism etc. are with a wide range of applications.
Compared with prior art, the present invention having the following advantages that and effect:
1. the present invention is prepared for the full-length infectious clone of M1 virus, have the characteristics that be easy to directional transformation M1 virus,
Convenient for carrying out relevant research.Establish the full-length infectious clone of M1 virus not yet both at home and abroad at present, the present invention has filled up this
Blank.
2. the present invention grinds the basic research (such as pathogenic mechanism, replicanism) and application of carrying out the virus of M1
Study carefully (such as neural circuitry label, infected tumor and killing, drug screening, Characterization of antigenic epitopes, new generation vaccine and diagnostic reagent)
It has important practical significance and wide application value.
Detailed description of the invention
Fig. 1 is a kind of building schematic diagram of M1 virus full length infection clones.
Fig. 2 is the schematic diagram that a kind of M1 virus full length infective cloned plasmids generate M1 virus.
Wherein, A: the cell of untransfected M1 virus full length infective cloned plasmids；
B: the cell of transfection M1 virus full length infective cloned plasmids；
Fig. 3 is that a kind of RNA of M1 virus full length infection clones generates the schematic diagram of M1 virus.
Wherein, A: the cell of untransfected M1 virus full-length genome RNA；
B: the cell of transfection M1 virus full-length genome RNA.
Specific embodiment
Technical solution of the present invention is if not otherwise specified the ordinary skill in the art
Embodiment 1:M1 virus full length infection clones, construction method are as follows:
1. the virus genomic complementary DNA of segmented synthesis M1 and carrier DNA: synthesis segment F1, F2, F3, F4 and F5；
The sequence of F1, F2, F3, F4 and F5 segment is respectively SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:
4, shown in SEQ ID NO:5；Contain in the carrier sequence F5 of synthesis: Ubc promoter sequence, T7 promoter sequence, hdvr sequence
Column, BGHpA sequence；
F1, F2, F3, F4 and F5 segment synthesized above is inserted into pUC57 respectively, the carrier of acquisition is respectively designated as:
PUC57-F1, pUC57-F2, pUC57-F3, pUC57-F4 and pUC57-F5.
The splicing of four segments of 2.M1 viral genome: using PacI and RsrII digestion pUC57-F1 and pUC57-F5 respectively,
Large fragment is separately recovered using the method that glue recycles, using Gibson Assembly Cloning Kit homologous recombination kit
F1 and F2 segment is inserted into pUC57-F5, recombinant products transformed competence colibacillus HB101, PCR are accredited as positive clone and carried out
It cultivates and extracts plasmid and be sequenced, it is M1-F1 that correct clone designation, which is sequenced,；Then PacI and XhoI digestion pUC57- is used
Large fragment is separately recovered using the method that glue recycles in F2 and M1-F1, the use of concentration is that 1% agarose gel electrophoresis detects DNA's
F2 segment is inserted into M1-F1 using Gibson Assembly Cloning Kit homologous recombination kit, will recombinated by quality
Product transformed competence colibacillus HB101, the clone that PCR is accredited as the positive, which cultivates and extracts plasmid, to be sequenced, and sequencing is correct
Clone designation is M1-F1-F2；Then PacI and BglII digestion pUC57-F3 and M1-F1-F2 is used, the method recycled using glue
Large fragment is separately recovered, the use of concentration is the quality that 1% agarose gel electrophoresis detects DNA, using Gibson Assembly
F3 segment is inserted into M1-F1-F2 by Cloning Kit homologous recombination kit, by recombinant products transformed competence colibacillus HB101, PCR
The clone for being accredited as the positive, which cultivates and extracts plasmid, to be sequenced, and it is M1-F1-F2-F3 that correct clone designation, which is sequenced,；So
PacI and BlpI digestion pUC57-F4 and M1-F1-F2-F3 is used afterwards, is separately recovered pUC57-F4's using the method that glue recycles
The large fragment of small fragment and M1-F1-F2-F3 is the quality that 1% agarose gel electrophoresis detects DNA using concentration, uses
F3 segment is inserted into M1-F1-F2-F3 by Gibson Assembly Cloning Kit homologous recombination kit, by recombinant products
Transformed competence colibacillus HB101, the clone that PCR is accredited as the positive, which cultivates and extracts plasmid, to be sequenced, and correctly clone is sequenced
It is named as M1-F1-F2-F3-F4, the full-length infectious clone pFLM1 of as M1 virus, sequence is shown in SEQ ID NO:6.
Embodiment 2: based on M1 virus full length infective cloned plasmids preparation M1 virus:
There is ubiquitin promoter due to being located at the virus genomic upstream M1 in pFLM1, M1 virus can be driven complete
The transcription of long genome, thus by the full-length infectious cloned plasmids pFLM1 (2 μ g) of pFLM1 transfect BHK21 cell, 37 DEG C,
5% (v/v) CO2It is cultivated in incubator, different time uses micro- sem observation cell state, and collects in virus in different time
Clearly；And the cell of untransfected plasmid is set simultaneously as a control group, pass through the cellular of microscope observation experiment group and control group
State.
(A in Fig. 2) occurs for the cell of untransfected pFLM1 plasmid, cell-free lesion, the observation transfection pFLM1 matter under light field
The BHK21 cell of grain, it is seen that apparent cytopathy (B in Fig. 2) shows to have produced recombinant virus.Above data shows
The full-length infectious clone of M1 virus has the ability for preparing M1 virus.
Embodiment 3: it is transcribed in vitro based on M1 virus full length infection clones as preparation M1 virus after RNA:
With MluI digestion pFLM1, the product being digested is recycled, uses MEGAscript T7Transcription Kit body
PFLM1 after outer transcription digestion becomes RNA, and transfects BHK21 cell, and 37 DEG C, 5% (v/v) CO2It is cultivated in incubator, and same
When setting untransfected RNA cell as a control group, pass through the cell state of microscope observation experiment group and control group.
(A in Fig. 3) occurs for the cell of untransfected M1 viral RNA, cell-free lesion, the observation transfection M1 virus under light field
The BHK21 cell of RNA, it is seen that apparent cytopathy (B in Fig. 3) shows to have produced recombinant virus.Above data shows
The full-length infectious clone of M1 virus has the ability for preparing M1 virus.
Experimental method in finally, it should also be noted that above-described embodiment is unless otherwise specified conventional method,
And the above list is only a few specific embodiments of the present invention.It, can be with it is clear that the invention is not restricted to above embodiments
There are many deformations.All changes that those skilled in the art directly can export or associate from present disclosure
Shape is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Wuhan Inst. of Physics and Mathematics, Chinese Academy of Sciences
<120>a kind of M1 virus full length infection clones and preparation method and its preparing M1 virus in application
<130>a kind of M1 virus full length infection clones and preparation method and its preparing M1 virus in application
<160> 6
<170> PatentIn version 3.1
<210> 1
<211> 3274
<212> DNA
<213>artificial sequence
<400> 1
ttaattaata cgacctgctc gaggccacga tgacgtgtaa caacagtgca cgccaccgtc 60
gcagtgtgac ggaacacttc aatgtctaca aggccacgaa accgtaccta gcgtattgcg 120
cggactgcgg agacgggcag ttctgttaca gcccggtggc tatagaaaaa attagggatg 180
aggcttccga tggcatgata aagatccagg tcgcagcgca aattggcatc aacaaaggag 240
gaacacacga acacaacaaa atcaggtaca tcgccgggca tgacatgaaa gaggcaaacc 300
gggattcttt acaagtgcat acttccggtg tgtgcgctat tcgaggcacg atgggccact 360
tcatcgtggc ctactgccct ccagggggcg aactaaaggt ccagttccaa gatgcagaat 420
cgcacacaca ggcctgcaaa gtgcagtaca aacacgctcc ggccccagta ggcagagaaa 480
aattcaccgt caggccccac ttcggtatcg aagtgccatg cacaacgtac cagctgacta 540
ccgcaccgac ggaggaagag atcgacatgc ataccccacc ggatatccca gacataacgt 600
tgctgtcgca gcagtcaggt aacgtaaaga tcacagcagg aggaaaaacc atcagataca 660
actgcacgtg tggtagtggc aacgtgggca ccaccagtag cgacaagact atcaattcgt 720
gcaaaatagc acagtgccac gctgcggtga ctaaccacga taagtggcag tacacctcct 780
cgtttgtccc tagagccgac cagttgtctc gcaaaggtaa agtgcacgta cccttccctc 840
tgaccaactc cacatgcagg gtgccccttg cacgtgcacc aggtgtcaca tacggaaaga 900
gagaactgac agtgaaactg catccagatc atcccacgct gttgacgtac cggagtctag 960
gagcagatcc gcgtccgtat gaggagtgga tagaccgata cgtcgaacgg accataccgg 1020
tgaccgaaga tgggatagag tacagatggg gaaacaaccc acccgtgcgc ttgtgggccc 1080
agctgacaac tgaaggcaaa ccccatgggt ggccgcacga gatcatactc tattactatg 1140
ggctataccc agcagccacc atcgtcgctg tctcagccgc gtgtctcgca gtcgtactat 1200
cgctgctggc gtcatgttac atgttcgcca ctgcacgccg caagtgcctg accccatacg 1260
ccctgacccc cggagccgtc gtcccggtaa cactaggagt actatgctgc gcaccacgag 1320
cgcatgccgc gtcgtttgcg gaatctatgg cgtatctatg ggatgagaat caaaccctgt 1380
tttggctgga gcttgcaacg ccgctcgctg ccataatcat acttgtatgc tgcctgaaga 1440
acctgctttg ctgctgcaaa ccgctatctt ttttagtgtt ggtgagcctg ggaactcccg 1500
tcgtaaaatc ttacgaacac accgcaacga tcccgaatgt ggtgggattc ccgtataagg 1560
ctcacattga gaggaacggc ttctccccga tgaccctaca gcttgaggta cttggaacca 1620
gcttggaacc cacactaaac ttagagtaca taacctgtga atacaagaca gtcgtgccgt 1680
caccttatat caagtgctgc gggacatcag aatgcagatc catggagcgc cccgactatc 1740
aatgccaggt ctacacagga gtgtacccat ttatgtgggg cggcgcatac tgcttctgcg 1800
acactgagaa cacccagctg agtgaagcat acgttgacag atcggacgta tgcaagcacg 1860
accatgccgc ggcctacaag gcgcatactg cggcaatgaa agccaccatc cgaataagct 1920
acgggaacct caatcagaca acaacggcgt tcgtcaacgg ggagcacaca gtgaccgtcg 1980
gaggcagcag gtttactttt ggtccaatct ccactgcctg gacgcctttc gacaacaaga 2040
ttgtcgtcta taagaacgac gtctacaacc tggacttccc accctacggg tcaggacaac 2100
caggaaggtt tggagacatc cagagcagga cggtagagag caaggacctg tatgccaaca 2160
ccgccctcaa gttgtcaaga ccttcgtccg gtactgttca cgtgccttac acacagaccc 2220
cttctggctt taagtactgg ataaaagaga gaggcacgtc gctgaatgac aaggctccct 2280
ttggatgcgt aatcaagacc aacccagtta gagcagaaaa ttgcgccgtt ggcaacatcc 2340
cagtctccat ggacatcccg gactccgcgt ttacgcgcgt gattgatgca cctgccgtca 2400
caaacctgga gtgccaagtg gcggtctgca cgcactcacc ggacttcggc gggattgcga 2460
ctctgacttt caaaaccgac aaacccggaa aatgtgctgt ccattctcat tcgaacgtag 2520
ccaccataca ggaggcagct gtggacatca aaacagatgg caagataacc ctgcatttct 2580
ctacagcatc agcatccccg gcattcaagg tatctgtgtg cagtgccaaa acgacatgca 2640
tggcagcgtg tgagccgccg aaggattaca tcgtccctta tggggcgagc cataacaacc 2700
aagtttttcc tgacatgtct ggcacggcaa tgacatgggt gcagcgggta gctggcggat 2760
tcggcgggct aacactcgcc gctgtggcag tacttatact ggtgacgtgt gtgactatgc 2820
gccgctaacc gggaggcttg acataatgta tatatataag catcatagtt ttaataaagc 2880
acataaataa tcaagtagat caaagggcta cctaacccct gaatagtaac aaaacgcaaa 2940
atacaaaaac attagttcaa agggccagta acccctgaat agtaacaaaa cataaaaacc 3000
aaaaaaaaac agtagttcaa agggctatac aacccctgaa tagtaacaaa atacagaaaa 3060
accataaaaa ttataaaatt aactaatctg atcatctaaa tttgactaat tggaaatagc 3120
cgaactctac ggagatgtag gcgtccgaac tccacggaga cgtaggacaa aattctgccg 3180
aaccccagac catcggggac gtaggcgtct aatttgtttt tttaatattt tacacgcgtg 3240
ggtcggcatg gcatctccac ctcctcgcgg tccg 3274
<210> 2
<211> 3749
<212> DNA
<213>artificial sequence
<400> 2
ttaattaagc taacgagcag atctgcctgt acgccctagg ggagacgatg gacagcatac 60
gcactaaatg cccagtagag gacgccgatt cgtctacgcc gccgaaaacg gtaccgtgtc 120
tatgtcggta cgcgatgacc gcggagcggg ttgccagact taggatgaat aacaccaaaa 180
acatcatcgt gtgctcctcc tttccattgc cgaagtacag gatagaaggc gtgcagaagg 240
tgaagtgtga ccgagtgcta atttttgacc agaccgtccc gtcactagta agtcccagaa 300
agtacataca gcagccgccg gaacagctgg ataatgtgag cctgacttct acgacgtcga 360
cgggatccgc atggtcattt ccatcggaaa cgacctacga aaccatggaa gtcgtagccg 420
aggtacacac cgaacctccg atccctccgc ctcgccgacg tagagcagcc gtcgcccaac 480
ttagacagga tctggaagtc accgaggaga tcgagccgta cgtgatacag caagcagaga 540
tcatggtcat ggagagggtc gcgacgacag acatacgcgc tatcccagtc ccggcacggc 600
gggccattac aatgccagtc ccagccccca gtgttcgtaa ggtcgctacc gaacctccat 660
tagaaccgga agctcctatc ccggcaccaa gaaagagaag aaccactggc accacacctc 720
cgcataaccc cgaggatttc gttcccaggg taccggttga gttaccgtgg gagccggaag 780
acctagacat ccaattcggt gacttggagc cacgccgccg gaacaccagg gaccgagatg 840
tcagcacagg aatacagttc ggtgacatcg actttaacca gtccagacta ggcagggctg 900
gcgcgtatat cttttcgtct gacactggcc cgggtcacct acagcagaag tccgtaaggc 960
aacatgaatt gccatgcgag actctgtatg cccatgaaga cgaacgcata tacccgccgg 1020
catttgacgg agagaaagag aaggtactcc aggcaaagat gcagatggcc ccgacagaag 1080
cgaataagag caggtaccag tcgaggaaag tagagaacat gaaggcatta attgtagaaa 1140
gactacgcga aggagcaaag atgtacctcc atgagcaaac cgacaaagta cccacgtaca 1200
ccagcaagta ccctagacct gtgtactcac catcggtgga tgacagcctg agcgatccgg 1260
aagtggctgt ggccgcctgt aactctttct tagaggagaa ttatccgacc gtggcgaact 1320
accagataac cgatgagtat gacgcttatc tggacttggt cgacggctct gaaagctgcc 1380
tcgacagagc tacgttctgc ccggccaaac taagatgtta ccctaagcac cacgcatacc 1440
accaaccaca aatcaggagc gcagtacctt cccctttcca aaacacgtta caaaacgtgc 1500
tagccgcggc cactaaaaga aattgtaatg tcacccaaat gagagaatta ccaaccatgg 1560
actctgcggt gtttaacgta gaaagcttca aaaaatacgc ctgtaccggc gaatattggc 1620
aagaatttaa agacaatcct atacggatca ccaccgaaaa cataacgacg tacgtggcta 1680
aactcaaggg tccaaaggct gctgcccttt ttgccaagac gcataacctg gtgccgctcc 1740
aggaggtgcc aatggaccgc ttcgtgatgg atatgaagag agatgtgaaa gttacaccag 1800
gcaccaagca taccgaagaa aggcctaaag tgcaagtgat tcaagcggcg gaaccattgg 1860
ccacggcata tttatgcgga atccacagag agttagtcag gcggctaaaa gccgttctga 1920
ccccgaacat tcacactctg tttgacatgt cggcggagga ctttgatgcc atcatagcgg 1980
cacattttca accgggagat gctgtactgg agacagatat cgcatccttc gataagagcc 2040
aagacgactc cttagcgcta acggcgctaa tgcttctgga agacctcggg gtcgaccaag 2100
aactgctgga ccttatcgaa gctgcgtttg gtgagatcac gggtgtgcat ctacctaccg 2160
gtacaagatt taaattcggt gctatgatga agtcaggaat gtttcttaca ctcttcatca 2220
acacgctgct gaacattgtc atagcgtgcc gcgtcttacg cgacaaatta tcgtcctcgg 2280
cgtgcgccgc cttcataggt gatgacaaca tagtgcacgg cgtgaggtca gacccgctaa 2340
tggcagaaag gtgtgcgagt tgggtcaaca tggaagtaaa gatcatcgat gccacaatgt 2400
gtgagaaacc accgtacttt tgtggaggat tcatcctgta cgacagtgtc gccggtacag 2460
cgtgtagggt tgcagacccg ttaaagaggc tgttcaaact cgggaaaccg ctcccagcgg 2520
acgacaacca ggatgaagac agaagaaggg cactaaaaga cgaaacagtt aagtggtccc 2580
gcataggatt gagagaagaa ttagacgtgg cactgagctc aagataccaa gtcagtggcg 2640
tcgggaacat cactagagcg atgtccacgc tgtctaagaa tttgaagtct tttaggaaaa 2700
taagaggtcc cattatacat ctgtacggcg gtcctaaata gatgcaggat tacactacat 2760
ctaaagacca cgtattacag acaccatgaa ttacatccca actcaaacct tttacggacg 2820
ccgttggcga ccacgcccgg cgttccgtcc atggcgggtg ccgatgcagc cggccccacc 2880
catgattcca gagctgcaaa ctccgatcgt ccaggcccaa cagatgcagc agctaatcag 2940
tgcagtttct gccctgacga ctaagcaaaa tggcaaagca ccgaagaagc cgaagaaaaa 3000
gccgcaaaaa gcgaaggcta agaaaaacga acagcaaaag aaaaacgaga acaagaaacc 3060
accacctaag cagaagaatc cggctaagaa gaagaaacca ggaaaaaggg aacgcatgtg 3120
catgaagata gaaaatgatt gcatcttcga ggtcaagctt gacggtaagg tcacgggata 3180
cgcctgccta gtcggggata aagtgatgaa gccggcacac gtcaaaggtg tgatcgacaa 3240
ccccgaccta gcgaagctta cctacaagaa atcgagcaag tatgacctag agtgcgccca 3300
gataccggtg cacatgaagt cagatgcttc aaaatacacc catgaaaaac cagaagggca 3360
ctacaattgg catcacggtg cagtgcagta cagcggtggc aggttcacaa tcccgacagg 3420
cgcaggtaaa ccaggagaca gcggccggcc gatcttcgac aacaaaggac gtgtggtggc 3480
cattgtcctg ggaggggcca acgaaggagc caggactgcc ctatctgtcg tgacctggac 3540
caaagacatg gtcacacggt acaccccaga aggaacagaa gaatggtccg ccgccttgat 3600
gatgtgcgtc ttagccaacg ttacattccc acgctcagag cccgcatgtg caccctgttg 3660
ctatgaaaaa caaccagaac agacactgag gatgttggag gacaacgtgg accgcccggg 3720
ctactacgac ctgctcgagg ccacgatga 3749
<210> 3
<211> 2981
<212> DNA
<213>artificial sequence
<400> 3
ttaattaacc accctcttgc tgagcaagtc acggtcatga cccactctgg aagatccggc 60
agatacccag tcgacaagta cgacggacgg gtattgatcc caacaggagc ggccatccca 120
gtgagtgagt tccaggcact cagcgagagc gcaaccatgg tgtacaatga gagggaattt 180
ataaatcgca agctacacca catagcgcta tacgggccag ccttgaatac cgacgaggaa 240
agctacgaaa aagtgagagc tgagagggca gagacagagt atgtgtttga cgtggacaag 300
aaggcatgta tcaagaagga ggaggcatca ggccttgtgt taacagggga cctaatcaat 360
ccacctttcc acgaattcgc atacgaagga ctcaagatcc gcccagcagc cccgtaccac 420
acgacgatca ttggtgtgtt tggcgttcca ggttcgggca agtcggctat cattaagaac 480
atggtgacga ctcgcgatct ggtggccagt ggaaagaagg agaactgcca agagatcatg 540
aatgatgtaa agaggcaacg cgggttggac gtgaccgcta ggaccgtcga ctcaatctta 600
ttgaatgggt gcaagaaagg cgtagaaaac ctttacgtcg atgaggcgtt cgcgtgtcac 660
tcgggcactt tgctagcgct catcgcgctg gtgagaccgt caggtaaggt agtactgtgc 720
ggcgacccca agcagtgtgg tttcttcaat ttgatgcaac tgaaggtgca ctataaccac 780
aacatttgta caagggtgct ccataagagc atctccagaa gatgcactct acctgttacg 840
gcgatcgtgt ccaccttgca ctaccaaggg aagatgagaa cgacgaaccg atgcaacacc 900
cccattcaga ttgacaccac cggttcttcc aaaccagcct caggagatat cgtgttaacg 960
tgcttccgcg gctgggtgaa gcaactgcaa atcgactatc gtggacacga ggtgatgtcc 1020
gcagctgctt cccagggtct gacaaggaaa ggcgtgtacg ccgtgagaca gaaagtgaac 1080
gaaaacccac tgtactcacc actgtcggag cacgtcaatg tgctgttgac ccgaactgaa 1140
aaccgactgg tgtggaagac actgtcgggt gacccgtgga taaaggtgtt aaccaatgtt 1200
ccacgtgggg atttcagtgc aactttggag gaatggcaag aagaacatga cggtatcatg 1260
agagtgttga acgagcgacc ggcggaggtt gatccattcc aaaacaaggc caaagtgtgc 1320
tgggcaaaat gtctggtgca agttcttgag acggccggaa tacgtatgac ggcagatgaa 1380
tggaacacca tcttggcttt cagagaggac agagcgtact caccagaagt cgctctcaac 1440
gagatttgca ctcgttacta cggcgttgac ctagacagcg gcctattctc agcgcagtca 1500
gtttccctct tttatgagaa caaccactgg gacaacaggc ccggaggacg catgtacggg 1560
ttcaaccatg aagtagccag gaaatatgca gccaggtttc catttctacg tggcaacatg 1620
aactcggggc tacaactaaa cgtccctgag aggaagctcc aaccttttag cgctgaatgc 1680
aatatagtcc catccaatcg tcggttaccg catgctctag tcacaagcta tcagcagtgc 1740
cgtggggaga gggtagagtg gttgctgaaa aagattccag gtcaccaaat gttacttgta 1800
agtgagtaca acctggtgat acctcacaaa agagtcttct ggattgcacc tccgcgggtg 1860
tcaggcgcgg accgcacgta cgacatggac ctagggttac ctatggatgc aggccgttac 1920
gatctggtat tcgtcaacat ccatactgag taccggcaac accactacca acaatgcgtc 1980
gaccattcaa tgcgtctgca gatgctggga ggggattcac tacacctgct tagaccagga 2040
ggctcgctgc tgatgagagc atatggttac gcagacagag tcagcgagat ggtggtgaca 2100
gccctggcta ggaaattctc ggcgttccgt gtcctgagac cggcgtgtgt gacgagcaac 2160
acagaagtgt tcctgctgtt ttctaacttt gataacggca gaagagcggt aaccttgcac 2220
caagctaacc agaaacttag ctcaatgtat gcctgcaacg gactgcacac tgctggctgt 2280
gcaccgtcat acagggtccg ccgcgcagat atatcaggac acggtgagga agcagtcgta 2340
aatgctgcca atgccaaggg taccgtgagc gatggagtgt gcagggcggt cgctaagaag 2400
tggccatcat ctttcaaagg ggctgcaact ccagtcggca cagccaagat gatccgcgca 2460
gatggcatga ccgtaatcca cgcagtggga ccgaacttct ccaccgtaac agaagccgaa 2520
ggggacagag agctagcggc cgcgtaccga gctgtggcta gcataattag taccaacaac 2580
ataaagagcg tcgcagtacc gctgctatcc acaggcacct tttccggcgg taaggacaga 2640
gtgatgcagt ccttgaacca cttattcacg gcattggatg ccaccgatgc agacgtggtt 2700
atctactgca gagataaaaa ctgggaaaag aagattcagg aagccatcga caggcggacg 2760
gcaatagagc tcgtatccga agacgtgacc ttggaaaccg atctggttag ggtacacccg 2820
gacagttgct tagtcggcag aaatggttac agtgcaactg acggtaaact gtactcttac 2880
cttgagggca cgaggttcca ccagacggcg gtcgacatgg ctgaaatatc aactttatgg 2940
ccaagactcc aagatgctaa cgagcagatc tgcctgtacg c 2981
<210> 4
<211> 1837
<212> DNA
<213>artificial sequence
<400> 4
ttaattaaat ggcggacgtg tgacatcacc gttcgctctt tctaggatcc tttgctactc 60
cacatagtga gagacaaaca acccaaatga aggtaaccgt ggacgttgag gctgatagcc 120
cattccttaa ggcccttcag aaggcgtttc ccgcctttga ggttgaatca cagcaggtca 180
caccgaatga ccatgctaac gctagagcat tttcgcatct ggctactaaa ctgattgagc 240
aagaggttcc aacaggcgtc accatcctgg acgtgggtag tgcacccgca aggaggttga 300
tgtctgacca cacctaccac tgcatctgcc ccatgaaaag tgcggaagac ccagagaggc 360
tggcgaatta cgctcgaaag ctggcgaaag catcggggac tgtgctagac aagaatgtgt 420
ccggaaagat aacggaccta caagacgtca tggccactcc agacttggaa tccccgactt 480
tttgcctgca cactgacgag acgtgccgca ctagggctga ggtcgctgtg taccaggacg 540
tatacgctgt gcacgcaccg acgtcactgt atcaccaggc catcaaaggt gtcaggacgg 600
cgtattggat tggattcgac accactccat tcatgttcga ggcactagcg ggcgcgtatc 660
ctgcgtactc gaccaactgg gcagatgagc aagtgctgca ggctcgtaac atcggcctgt 720
gcgcgacagg cctctccgag gggcgtcgcg gcaaactctc catcatgaga aagaagtgct 780
tgcgaccgag cgacagagta atgttttcgg tcgggtccac cttgtacacc gagagccgaa 840
agctgctgcg cagctggcat ttaccttccg tgtttcacct gaagggtaag aacagtttta 900
cctgcaggtg cgacacggtg gtgtcatgcg aaggttacgt ggtaaagaag atcaccataa 960
gcccgggcat atatggaaaa acagtcgatt acgcagttac ccatcacgca gagggttttc 1020
tggtgtgtaa gatcactgat acagtcagag gagaaagagt ctctttcccg gtctgtacct 1080
atgtgcctgc aaccatatgc gatcagatga cgggtatact tgccaccgac gtgacaccag 1140
aggatgccca gaagctcctg gttggattga accaacgcat agtggtgaat ggtaggacgc 1200
aaagaaacac aaacacaatg aaaaactatc tactgccagt ggtagcgcaa gcattcagta 1260
aatgggcacg agaggcacgc gcagacatgg aggatgaaaa acccctaggc accagagaac 1320
gcacgttgac gtgttgttgc ctgtgggcgt ttaaaagcca caaaacccac accatgtata 1380
agcggcctga aactcaaact atcgtcaaag tgccttccac ttttgactcc tttgtgatac 1440
cgagcctgag gtcatccagt ctttccatgg atatcagacg gaggatcaaa ctgctactca 1500
gcgcaagaat ggcccaaggc ctaccatact caggagaccg cactgaagct cgcgcggcag 1560
aagaagaaga gaaggaggcg caggaggcag aacttacgag ggcagcgctg ccaccgctag 1620
tgagcggctc ttgtgctgac gatatcgccc aggtggatgt agaggaatta accttcagag 1680
ccggagccgg ggttgtggaa acacccagga atgccctgaa ggttacaccg caagcacacg 1740
accacctcat aggctcctac ttgatccttt ccccccaaac ggtgttgaaa agcgagaagc 1800
tggcacccat ccaccctctt gctgagcaag tcacggt 1837
<210> 5
<211> 4431
<212> DNA
<213>artificial sequence
<400> 5
gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc 60
agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg 120
ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga 180
cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta 240
gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata 300
taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt 360
cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg 420
gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc 480
tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa 540
atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt 600
gaaacaaggt ggggggcatg gtgggcggca agaacccaag gtcttgaggc cttcgctaat 660
gcgggaaagc tcttattcgg gtgagatggg ctggggcacc atctggggac cctgacgtga 720
agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg 780
gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt 840
cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg 900
cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg 960
cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat 1020
gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga 1080
gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta 1140
attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt 1200
tagactaata cgactcacta tagttaatta agggtcggca tggcatctcc acctcctcgc 1260
ggtccgacct gggctacttc ggtaggctaa gggagaagaa cttgtttatt gcagcttata 1320
atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 1380
attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctat ttaaatctgt 1440
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 1500
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 1560
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 1620
agacaatagc aggcatgctg gggatgcggt gggctctatg gtcgcgattt ctagtctgca 1680
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 1740
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 1800
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 1860
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 1920
gctccgcccc cctgacgagc atcacaaaaa tcgaccctca agtcaaaggg ggcgaaaccc 1980
gacaggacta taaaaatacc aggcgtttcc ccctggaagc tccctcgggc gctctcctgt 2040
tccaaccctg ccgcttaccg gatacctgtc cccctttctc ccttcgggaa gcggggcgct 2100
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2160
ctgtgtgcac aaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 2220
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2280
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2340
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2400
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2460
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2520
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 2580
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 2640
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 2700
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 2760
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 2820
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 2880
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 2940
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3000
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3060
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3120
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3180
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3240
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 3300
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3360
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3420
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 3480
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 3540
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 3600
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 3660
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 3720
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 3780
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 3840
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 3900
actgagagtg caccattcga cgctctccct tatgcgactc ctgcattagg aagcagccca 3960
gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg 4020
cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca 4080
tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 4140
caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatctggc 4200
tagcgatgac cctgctgatt ggttcgctga ccatttccgg gtgcgggacg gcgttaccag 4260
aaactcagaa ggttcgtcca accaaaccga ctctgacggc agtttacgag agagatgata 4320
gggtctgctt cagtaagcca gatgctacac aattaggctt gtacatattg tcgttagaac 4380
gcggctacaa ttaatacata accttatgta tcatacacat acgggcgcgc c 4431
<210> 6
<211> 16127
<212> DNA
<213>artificial sequence
<400> 6
gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc 60
agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg 120
ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga 180
cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta 240
gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata 300
taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt 360
cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg 420
gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc 480
tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa 540
atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt 600
gaaacaaggt ggggggcatg gtgggcggca agaacccaag gtcttgaggc cttcgctaat 660
gcgggaaagc tcttattcgg gtgagatggg ctggggcacc atctggggac cctgacgtga 720
agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg 780
gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt 840
cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg 900
cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg 960
cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat 1020
gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga 1080
gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta 1140
attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt 1200
tagactaata cgactcacta tagttaatta aatggcggac gtgtgacatc accgttcgct 1260
ctttctagga tcctttgcta ctccacatag tgagagacaa acaacccaaa tgaaggtaac 1320
cgtggacgtt gaggctgata gcccattcct taaggccctt cagaaggcgt ttcccgcctt 1380
tgaggttgaa tcacagcagg tcacaccgaa tgaccatgct aacgctagag cattttcgca 1440
tctggctact aaactgattg agcaagaggt tccaacaggc gtcaccatcc tggacgtggg 1500
tagtgcaccc gcaaggaggt tgatgtctga ccacacctac cactgcatct gccccatgaa 1560
aagtgcggaa gacccagaga ggctggcgaa ttacgctcga aagctggcga aagcatcggg 1620
gactgtgcta gacaagaatg tgtccggaaa gataacggac ctacaagacg tcatggccac 1680
tccagacttg gaatccccga ctttttgcct gcacactgac gagacgtgcc gcactagggc 1740
tgaggtcgct gtgtaccagg acgtatacgc tgtgcacgca ccgacgtcac tgtatcacca 1800
ggccatcaaa ggtgtcagga cggcgtattg gattggattc gacaccactc cattcatgtt 1860
cgaggcacta gcgggcgcgt atcctgcgta ctcgaccaac tgggcagatg agcaagtgct 1920
gcaggctcgt aacatcggcc tgtgcgcgac aggcctctcc gaggggcgtc gcggcaaact 1980
ctccatcatg agaaagaagt gcttgcgacc gagcgacaga gtaatgtttt cggtcgggtc 2040
caccttgtac accgagagcc gaaagctgct gcgcagctgg catttacctt ccgtgtttca 2100
cctgaagggt aagaacagtt ttacctgcag gtgcgacacg gtggtgtcat gcgaaggtta 2160
cgtggtaaag aagatcacca taagcccggg catatatgga aaaacagtcg attacgcagt 2220
tacccatcac gcagagggtt ttctggtgtg taagatcact gatacagtca gaggagaaag 2280
agtctctttc ccggtctgta cctatgtgcc tgcaaccata tgcgatcaga tgacgggtat 2340
acttgccacc gacgtgacac cagaggatgc ccagaagctc ctggttggat tgaaccaacg 2400
catagtggtg aatggtagga cgcaaagaaa cacaaacaca atgaaaaact atctactgcc 2460
agtggtagcg caagcattca gtaaatgggc acgagaggca cgcgcagaca tggaggatga 2520
aaaaccccta ggcaccagag aacgcacgtt gacgtgttgt tgcctgtggg cgtttaaaag 2580
ccacaaaacc cacaccatgt ataagcggcc tgaaactcaa actatcgtca aagtgccttc 2640
cacttttgac tcctttgtga taccgagcct gaggtcatcc agtctttcca tggatatcag 2700
acggaggatc aaactgctac tcagcgcaag aatggcccaa ggcctaccat actcaggaga 2760
ccgcactgaa gctcgcgcgg cagaagaaga agagaaggag gcgcaggagg cagaacttac 2820
gagggcagcg ctgccaccgc tagtgagcgg ctcttgtgct gacgatatcg cccaggtgga 2880
tgtagaggaa ttaaccttca gagccggagc cggggttgtg gaaacaccca ggaatgccct 2940
gaaggttaca ccgcaagcac acgaccacct cataggctcc tacttgatcc tttcccccca 3000
aacggtgttg aaaagcgaga agctggcacc catccaccct cttgctgagc aagtcacggt 3060
catgacccac tctggaagat ccggcagata cccagtcgac aagtacgacg gacgggtatt 3120
gatcccaaca ggagcggcca tcccagtgag tgagttccag gcactcagcg agagcgcaac 3180
catggtgtac aatgagaggg aatttataaa tcgcaagcta caccacatag cgctatacgg 3240
gccagccttg aataccgacg aggaaagcta cgaaaaagtg agagctgaga gggcagagac 3300
agagtatgtg tttgacgtgg acaagaaggc atgtatcaag aaggaggagg catcaggcct 3360
tgtgttaaca ggggacctaa tcaatccacc tttccacgaa ttcgcatacg aaggactcaa 3420
gatccgccca gcagccccgt accacacgac gatcattggt gtgtttggcg ttccaggttc 3480
gggcaagtcg gctatcatta agaacatggt gacgactcgc gatctggtgg ccagtggaaa 3540
gaaggagaac tgccaagaga tcatgaatga tgtaaagagg caacgcgggt tggacgtgac 3600
cgctaggacc gtcgactcaa tcttattgaa tgggtgcaag aaaggcgtag aaaaccttta 3660
cgtcgatgag gcgttcgcgt gtcactcggg cactttgcta gcgctcatcg cgctggtgag 3720
accgtcaggt aaggtagtac tgtgcggcga ccccaagcag tgtggtttct tcaatttgat 3780
gcaactgaag gtgcactata accacaacat ttgtacaagg gtgctccata agagcatctc 3840
cagaagatgc actctacctg ttacggcgat cgtgtccacc ttgcactacc aagggaagat 3900
gagaacgacg aaccgatgca acacccccat tcagattgac accaccggtt cttccaaacc 3960
agcctcagga gatatcgtgt taacgtgctt ccgcggctgg gtgaagcaac tgcaaatcga 4020
ctatcgtgga cacgaggtga tgtccgcagc tgcttcccag ggtctgacaa ggaaaggcgt 4080
gtacgccgtg agacagaaag tgaacgaaaa cccactgtac tcaccactgt cggagcacgt 4140
caatgtgctg ttgacccgaa ctgaaaaccg actggtgtgg aagacactgt cgggtgaccc 4200
gtggataaag gtgttaacca atgttccacg tggggatttc agtgcaactt tggaggaatg 4260
gcaagaagaa catgacggta tcatgagagt gttgaacgag cgaccggcgg aggttgatcc 4320
attccaaaac aaggccaaag tgtgctgggc aaaatgtctg gtgcaagttc ttgagacggc 4380
cggaatacgt atgacggcag atgaatggaa caccatcttg gctttcagag aggacagagc 4440
gtactcacca gaagtcgctc tcaacgagat ttgcactcgt tactacggcg ttgacctaga 4500
cagcggccta ttctcagcgc agtcagtttc cctcttttat gagaacaacc actgggacaa 4560
caggcccgga ggacgcatgt acgggttcaa ccatgaagta gccaggaaat atgcagccag 4620
gtttccattt ctacgtggca acatgaactc ggggctacaa ctaaacgtcc ctgagaggaa 4680
gctccaacct tttagcgctg aatgcaatat agtcccatcc aatcgtcggt taccgcatgc 4740
tctagtcaca agctatcagc agtgccgtgg ggagagggta gagtggttgc tgaaaaagat 4800
tccaggtcac caaatgttac ttgtaagtga gtacaacctg gtgatacctc acaaaagagt 4860
cttctggatt gcacctccgc gggtgtcagg cgcggaccgc acgtacgaca tggacctagg 4920
gttacctatg gatgcaggcc gttacgatct ggtattcgtc aacatccata ctgagtaccg 4980
gcaacaccac taccaacaat gcgtcgacca ttcaatgcgt ctgcagatgc tgggagggga 5040
ttcactacac ctgcttagac caggaggctc gctgctgatg agagcatatg gttacgcaga 5100
cagagtcagc gagatggtgg tgacagccct ggctaggaaa ttctcggcgt tccgtgtcct 5160
gagaccggcg tgtgtgacga gcaacacaga agtgttcctg ctgttttcta actttgataa 5220
cggcagaaga gcggtaacct tgcaccaagc taaccagaaa cttagctcaa tgtatgcctg 5280
caacggactg cacactgctg gctgtgcacc gtcatacagg gtccgccgcg cagatatatc 5340
aggacacggt gaggaagcag tcgtaaatgc tgccaatgcc aagggtaccg tgagcgatgg 5400
agtgtgcagg gcggtcgcta agaagtggcc atcatctttc aaaggggctg caactccagt 5460
cggcacagcc aagatgatcc gcgcagatgg catgaccgta atccacgcag tgggaccgaa 5520
cttctccacc gtaacagaag ccgaagggga cagagagcta gcggccgcgt accgagctgt 5580
ggctagcata attagtacca acaacataaa gagcgtcgca gtaccgctgc tatccacagg 5640
caccttttcc ggcggtaagg acagagtgat gcagtccttg aaccacttat tcacggcatt 5700
ggatgccacc gatgcagacg tggttatcta ctgcagagat aaaaactggg aaaagaagat 5760
tcaggaagcc atcgacaggc ggacggcaat agagctcgta tccgaagacg tgaccttgga 5820
aaccgatctg gttagggtac acccggacag ttgcttagtc ggcagaaatg gttacagtgc 5880
aactgacggt aaactgtact cttaccttga gggcacgagg ttccaccaga cggcggtcga 5940
catggctgaa atatcaactt tatggccaag actccaagat gctaacgagc agatctgcct 6000
gtacgcccta ggggagacga tggacagcat acgcactaaa tgcccagtag aggacgccga 6060
ttcgtctacg ccgccgaaaa cggtaccgtg tctatgtcgg tacgcgatga ccgcggagcg 6120
ggttgccaga cttaggatga ataacaccaa aaacatcatc gtgtgctcct cctttccatt 6180
gccgaagtac aggatagaag gcgtgcagaa ggtgaagtgt gaccgagtgc taatttttga 6240
ccagaccgtc ccgtcactag taagtcccag aaagtacata cagcagccgc cggaacagct 6300
ggataatgtg agcctgactt ctacgacgtc gacgggatcc gcatggtcat ttccatcgga 6360
aacgacctac gaaaccatgg aagtcgtagc cgaggtacac accgaacctc cgatccctcc 6420
gcctcgccga cgtagagcag ccgtcgccca acttagacag gatctggaag tcaccgagga 6480
gatcgagccg tacgtgatac agcaagcaga gatcatggtc atggagaggg tcgcgacgac 6540
agacatacgc gctatcccag tcccggcacg gcgggccatt acaatgccag tcccagcccc 6600
cagtgttcgt aaggtcgcta ccgaacctcc attagaaccg gaagctccta tcccggcacc 6660
aagaaagaga agaaccactg gcaccacacc tccgcataac cccgaggatt tcgttcccag 6720
ggtaccggtt gagttaccgt gggagccgga agacctagac atccaattcg gtgacttgga 6780
gccacgccgc cggaacacca gggaccgaga tgtcagcaca ggaatacagt tcggtgacat 6840
cgactttaac cagtccagac taggcagggc tggcgcgtat atcttttcgt ctgacactgg 6900
cccgggtcac ctacagcaga agtccgtaag gcaacatgaa ttgccatgcg agactctgta 6960
tgcccatgaa gacgaacgca tatacccgcc ggcatttgac ggagagaaag agaaggtact 7020
ccaggcaaag atgcagatgg ccccgacaga agcgaataag agcaggtacc agtcgaggaa 7080
agtagagaac atgaaggcat taattgtaga aagactacgc gaaggagcaa agatgtacct 7140
ccatgagcaa accgacaaag tacccacgta caccagcaag taccctagac ctgtgtactc 7200
accatcggtg gatgacagcc tgagcgatcc ggaagtggct gtggccgcct gtaactcttt 7260
cttagaggag aattatccga ccgtggcgaa ctaccagata accgatgagt atgacgctta 7320
tctggacttg gtcgacggct ctgaaagctg cctcgacaga gctacgttct gcccggccaa 7380
actaagatgt taccctaagc accacgcata ccaccaacca caaatcagga gcgcagtacc 7440
ttcccctttc caaaacacgt tacaaaacgt gctagccgcg gccactaaaa gaaattgtaa 7500
tgtcacccaa atgagagaat taccaaccat ggactctgcg gtgtttaacg tagaaagctt 7560
caaaaaatac gcctgtaccg gcgaatattg gcaagaattt aaagacaatc ctatacggat 7620
caccaccgaa aacataacga cgtacgtggc taaactcaag ggtccaaagg ctgctgccct 7680
ttttgccaag acgcataacc tggtgccgct ccaggaggtg ccaatggacc gcttcgtgat 7740
ggatatgaag agagatgtga aagttacacc aggcaccaag cataccgaag aaaggcctaa 7800
agtgcaagtg attcaagcgg cggaaccatt ggccacggca tatttatgcg gaatccacag 7860
agagttagtc aggcggctaa aagccgttct gaccccgaac attcacactc tgtttgacat 7920
gtcggcggag gactttgatg ccatcatagc ggcacatttt caaccgggag atgctgtact 7980
ggagacagat atcgcatcct tcgataagag ccaagacgac tccttagcgc taacggcgct 8040
aatgcttctg gaagacctcg gggtcgacca agaactgctg gaccttatcg aagctgcgtt 8100
tggtgagatc acgggtgtgc atctacctac cggtacaaga tttaaattcg gtgctatgat 8160
gaagtcagga atgtttctta cactcttcat caacacgctg ctgaacattg tcatagcgtg 8220
ccgcgtctta cgcgacaaat tatcgtcctc ggcgtgcgcc gccttcatag gtgatgacaa 8280
catagtgcac ggcgtgaggt cagacccgct aatggcagaa aggtgtgcga gttgggtcaa 8340
catggaagta aagatcatcg atgccacaat gtgtgagaaa ccaccgtact tttgtggagg 8400
attcatcctg tacgacagtg tcgccggtac agcgtgtagg gttgcagacc cgttaaagag 8460
gctgttcaaa ctcgggaaac cgctcccagc ggacgacaac caggatgaag acagaagaag 8520
ggcactaaaa gacgaaacag ttaagtggtc ccgcatagga ttgagagaag aattagacgt 8580
ggcactgagc tcaagatacc aagtcagtgg cgtcgggaac atcactagag cgatgtccac 8640
gctgtctaag aatttgaagt cttttaggaa aataagaggt cccattatac atctgtacgg 8700
cggtcctaaa tagatgcagg attacactac atctaaagac cacgtattac agacaccatg 8760
aattacatcc caactcaaac cttttacgga cgccgttggc gaccacgccc ggcgttccgt 8820
ccatggcggg tgccgatgca gccggcccca cccatgattc cagagctgca aactccgatc 8880
gtccaggccc aacagatgca gcagctaatc agtgcagttt ctgccctgac gactaagcaa 8940
aatggcaaag caccgaagaa gccgaagaaa aagccgcaaa aagcgaaggc taagaaaaac 9000
gaacagcaaa agaaaaacga gaacaagaaa ccaccaccta agcagaagaa tccggctaag 9060
aagaagaaac caggaaaaag ggaacgcatg tgcatgaaga tagaaaatga ttgcatcttc 9120
gaggtcaagc ttgacggtaa ggtcacggga tacgcctgcc tagtcgggga taaagtgatg 9180
aagccggcac acgtcaaagg tgtgatcgac aaccccgacc tagcgaagct tacctacaag 9240
aaatcgagca agtatgacct agagtgcgcc cagataccgg tgcacatgaa gtcagatgct 9300
tcaaaataca cccatgaaaa accagaaggg cactacaatt ggcatcacgg tgcagtgcag 9360
tacagcggtg gcaggttcac aatcccgaca ggcgcaggta aaccaggaga cagcggccgg 9420
ccgatcttcg acaacaaagg acgtgtggtg gccattgtcc tgggaggggc caacgaagga 9480
gccaggactg ccctatctgt cgtgacctgg accaaagaca tggtcacacg gtacacccca 9540
gaaggaacag aagaatggtc cgccgccttg atgatgtgcg tcttagccaa cgttacattc 9600
ccacgctcag agcccgcatg tgcaccctgt tgctatgaaa aacaaccaga acagacactg 9660
aggatgttgg aggacaacgt ggaccgcccg ggctactacg acctgctcga ggccacgatg 9720
acgtgtaaca acagtgcacg ccaccgtcgc agtgtgacgg aacacttcaa tgtctacaag 9780
gccacgaaac cgtacctagc gtattgcgcg gactgcggag acgggcagtt ctgttacagc 9840
ccggtggcta tagaaaaaat tagggatgag gcttccgatg gcatgataaa gatccaggtc 9900
gcagcgcaaa ttggcatcaa caaaggagga acacacgaac acaacaaaat caggtacatc 9960
gccgggcatg acatgaaaga ggcaaaccgg gattctttac aagtgcatac ttccggtgtg 10020
tgcgctattc gaggcacgat gggccacttc atcgtggcct actgccctcc agggggcgaa 10080
ctaaaggtcc agttccaaga tgcagaatcg cacacacagg cctgcaaagt gcagtacaaa 10140
cacgctccgg ccccagtagg cagagaaaaa ttcaccgtca ggccccactt cggtatcgaa 10200
gtgccatgca caacgtacca gctgactacc gcaccgacgg aggaagagat cgacatgcat 10260
accccaccgg atatcccaga cataacgttg ctgtcgcagc agtcaggtaa cgtaaagatc 10320
acagcaggag gaaaaaccat cagatacaac tgcacgtgtg gtagtggcaa cgtgggcacc 10380
accagtagcg acaagactat caattcgtgc aaaatagcac agtgccacgc tgcggtgact 10440
aaccacgata agtggcagta cacctcctcg tttgtcccta gagccgacca gttgtctcgc 10500
aaaggtaaag tgcacgtacc cttccctctg accaactcca catgcagggt gccccttgca 10560
cgtgcaccag gtgtcacata cggaaagaga gaactgacag tgaaactgca tccagatcat 10620
cccacgctgt tgacgtaccg gagtctagga gcagatccgc gtccgtatga ggagtggata 10680
gaccgatacg tcgaacggac cataccggtg accgaagatg ggatagagta cagatgggga 10740
aacaacccac ccgtgcgctt gtgggcccag ctgacaactg aaggcaaacc ccatgggtgg 10800
ccgcacgaga tcatactcta ttactatggg ctatacccag cagccaccat cgtcgctgtc 10860
tcagccgcgt gtctcgcagt cgtactatcg ctgctggcgt catgttacat gttcgccact 10920
gcacgccgca agtgcctgac cccatacgcc ctgacccccg gagccgtcgt cccggtaaca 10980
ctaggagtac tatgctgcgc accacgagcg catgccgcgt cgtttgcgga atctatggcg 11040
tatctatggg atgagaatca aaccctgttt tggctggagc ttgcaacgcc gctcgctgcc 11100
ataatcatac ttgtatgctg cctgaagaac ctgctttgct gctgcaaacc gctatctttt 11160
ttagtgttgg tgagcctggg aactcccgtc gtaaaatctt acgaacacac cgcaacgatc 11220
ccgaatgtgg tgggattccc gtataaggct cacattgaga ggaacggctt ctccccgatg 11280
accctacagc ttgaggtact tggaaccagc ttggaaccca cactaaactt agagtacata 11340
acctgtgaat acaagacagt cgtgccgtca ccttatatca agtgctgcgg gacatcagaa 11400
tgcagatcca tggagcgccc cgactatcaa tgccaggtct acacaggagt gtacccattt 11460
atgtggggcg gcgcatactg cttctgcgac actgagaaca cccagctgag tgaagcatac 11520
gttgacagat cggacgtatg caagcacgac catgccgcgg cctacaaggc gcatactgcg 11580
gcaatgaaag ccaccatccg aataagctac gggaacctca atcagacaac aacggcgttc 11640
gtcaacgggg agcacacagt gaccgtcgga ggcagcaggt ttacttttgg tccaatctcc 11700
actgcctgga cgcctttcga caacaagatt gtcgtctata agaacgacgt ctacaacctg 11760
gacttcccac cctacgggtc aggacaacca ggaaggtttg gagacatcca gagcaggacg 11820
gtagagagca aggacctgta tgccaacacc gccctcaagt tgtcaagacc ttcgtccggt 11880
actgttcacg tgccttacac acagacccct tctggcttta agtactggat aaaagagaga 11940
ggcacgtcgc tgaatgacaa ggctcccttt ggatgcgtaa tcaagaccaa cccagttaga 12000
gcagaaaatt gcgccgttgg caacatccca gtctccatgg acatcccgga ctccgcgttt 12060
acgcgcgtga ttgatgcacc tgccgtcaca aacctggagt gccaagtggc ggtctgcacg 12120
cactcaccgg acttcggcgg gattgcgact ctgactttca aaaccgacaa acccggaaaa 12180
tgtgctgtcc attctcattc gaacgtagcc accatacagg aggcagctgt ggacatcaaa 12240
acagatggca agataaccct gcatttctct acagcatcag catccccggc attcaaggta 12300
tctgtgtgca gtgccaaaac gacatgcatg gcagcgtgtg agccgccgaa ggattacatc 12360
gtcccttatg gggcgagcca taacaaccaa gtttttcctg acatgtctgg cacggcaatg 12420
acatgggtgc agcgggtagc tggcggattc ggcgggctaa cactcgccgc tgtggcagta 12480
cttatactgg tgacgtgtgt gactatgcgc cgctaaccgg gaggcttgac ataatgtata 12540
tatataagca tcatagtttt aataaagcac ataaataatc aagtagatca aagggctacc 12600
taacccctga atagtaacaa aacgcaaaat acaaaaacat tagttcaaag ggccagtaac 12660
ccctgaatag taacaaaaca taaaaaccaa aaaaaaacag tagttcaaag ggctatacaa 12720
cccctgaata gtaacaaaat acagaaaaac cataaaaatt ataaaattaa ctaatctgat 12780
catctaaatt tgactaattg gaaatagccg aactctacgg agatgtaggc gtccgaactc 12840
cacggagacg taggacaaaa ttctgccgaa ccccagacca tcggggacgt aggcgtctaa 12900
tttgtttttt taatatttta cacgcgtggg tcggcatggc atctccacct cctcgcggtc 12960
cgacctgggc tacttcggta ggctaaggga gaagaacttg tttattgcag cttataatgg 13020
ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 13080
tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctatttaa atctgtgcct 13140
tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 13200
gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 13260
tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 13320
aatagcaggc atgctgggga tgcggtgggc tctatggtcg cgatttctag tctgcattaa 13380
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 13440
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 13500
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 13560
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 13620
cgcccccctg acgagcatca caaaaatcga ccctcaagtc aaagggggcg aaacccgaca 13680
ggactataaa aataccaggc gtttccccct ggaagctccc tcgggcgctc tcctgttcca 13740
accctgccgc ttaccggata cctgtccccc tttctccctt cgggaagcgg ggcgctttct 13800
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 13860
gtgcacaaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 13920
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 13980
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 14040
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 14100
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 14160
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 14220
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 14280
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 14340
atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 14400
gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 14460
atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 14520
ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 14580
cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 14640
agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 14700
cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 14760
tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 14820
agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 14880
gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 14940
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 15000
ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 15060
tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 15120
tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 15180
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 15240
caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 15300
atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 15360
gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 15420
tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 15480
acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 15540
gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 15600
agagtgcacc attcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 15660
taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc 15720
caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag 15780
cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 15840
cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tctggctagc 15900
gatgaccctg ctgattggtt cgctgaccat ttccgggtgc gggacggcgt taccagaaac 15960
tcagaaggtt cgtccaacca aaccgactct gacggcagtt tacgagagag atgatagggt 16020
ctgcttcagt aagccagatg ctacacaatt aggcttgtac atattgtcgt tagaacgcgg 16080
ctacaattaa tacataacct tatgtatcat acacatacgg gcgcgcc 16127
Claims (5)
1. a kind of M1 virus full length infection clones, sequence is shown in SEQ ID NO.6.
2. a kind of construction method of M1 virus full length infection clones uses the method for digestion and homologous recombination by M1 virus base
Because group four segments F1, F2, F3 and F4 are sequentially inserted between PacI the and RsrII restriction enzyme site of carrier F5, the sequencing of acquisition is just
True clone designation is M1-F1-F2-F3-F4, the full-length infectious clone pFLM1 of as M1 virus；
Described, the nucleotides sequence of F1 is classified as shown in SEQ ID NO:1, and the nucleotides sequence of F2 is classified as shown in SEQ ID NO:2, F3
Nucleotides sequence be classified as shown in SEQ ID NO:3, the nucleotides sequence of F4 is classified as shown in SEQ ID NO:4, the nucleotide sequence of F5
For shown in SEQ ID NO:5.
3. the application in the full-length infectious M1 virus for being cloned in preparation described in claim 1.
4. application according to claim 3, the full-length infectious clone is based on plasmid-transfected cells preparation M1 virus.
5. application according to claim 3, after in-vitro transcription of the full-length infectious clone based on promoter is RNA
Preparation M1 is viral after transfecting cell.
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