CN112877358A - Recombinant plasmid based on type 5 adenovirus sequence and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a recombinant plasmid based on a type 5 adenovirus sequence and application thereof. The recombinant plasmid of the invention is subjected to sequence optimization on the basis of a commonly used pHelper (11K) plasmid, Adv5 is used for replacing Adv2, and the recombinant plasmid is co-transfected with pRC and pAAV-GOI (gene of interest, recombinant plasmid carrying a target gene sequence) plasmid in HEK293 cells, so that the packaging amount of viruses is improved by more than 5 times compared with the commonly used pHelper plasmid. The plasmid can be applied to scientific research and large-scale adeno-associated virus preparation, and the production efficiency of adeno-associated viruses is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant plasmid based on a type 5 adenovirus sequence and application of the recombinant plasmid in-vitro preparation of adeno-associated viruses.
Background
AAV (Adeno-associated viruses) Adeno-associated viruses, which are derived from the family of parvoviridae (paravirus family), are single-stranded linear DNA-deficient viruses that usually require the assistance of helper viruses such as adenovirus and herpes virus to achieve self-replication (Buller, & H.1999; Atchison et al, 1965; Buller et al, 1981). While the adeno-associated virus vector system is a safe system for gene delivery in vivo and in vitro because it lacks replication, and has the characteristics of good safety, wide host cell range and long expression time in vivo (Samulski et al, 1989; Bueler, & H.1999). the current scientific community has a consensus that AAV does not cause human diseases (Berns and Linden, 1995).
The present preparation of adeno-associated virus can either use adenovirus or herpes simplex virus HSV directly to provide helper genes or use a three-plasmid cotransformation system. Three-plasmid cotransformation can reduce the risk of helper virus residues in the product. At present, a recombinant AAV (rAAV) System, namely an AAV Helper-Free System, is widely used in scientific research and production, and the System consists of three plasmids, namely pAAV-GOI, pRC vector and pHelper vector. The pAAV-GOI plasmid contains the gene of interest (GOI) and AAV Inverted Terminal Repeats (ITRs). The pRC vector plasmid contains AAV-encoded replication protein (rep) and viral capsid protein (cap) genes, which determine AAV serotype V (Hermonat et al, 1984; Ni et al, 1994). The pHelper vector plasmids provide the adenoviral gene products required for the production of infectious AAV viral particles, such as the E1B (Richardson and Westphal, 1984; Samulski and Shenk,1988), E2A (Jay et al, 1981; Janik et al, 1989), E4(Richardson and Westphal, 1981; Carter et al, 1983) and VA RNA (West et al, 1987; Janik et al, 1989) genes. The remaining adenoviral gene product is provided by a host cell that stably expresses the adenoviral E1 gene. The packaging system has the greatest advantages that auxiliary viruses are not added, so that on one hand, the pollution of the auxiliary viruses to the rAAV is eliminated; on the other hand, the possibility of generating wild type AAV is reduced, and the safety of AAV products is further improved.
Now widely used in recombinant AAV (rAAV) system is pHelper (11K) plasmid (https:// www.ncbi.nlm.nih.gov/nuccore/AF369965.1/), which is a helper plasmid with a full length of 11635bp and carrying ampicillin resistance gene, and the functional gene sequence of the plasmid is derived from adv 2. The plasmid consists of an Adv 2E 2A gene, and the nucleic acid position is 1-5336 bp; the adv 2E 4 gene with the nucleic acid position of 5337-8537 bp; adv2 VA gene with the nucleic acid position of 8538-9280 bp; pUC replication initiation at a nucleic acid position of 9367-10034 bp; ampicillin resistance gene, 10182-11042 bp; the ss-DNA replication origin f1, 11305-11600 bp.
Reference documents:
(1)Büeler,H.Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy[J].Biological Chemistry,1999,380(6):613-622.
(2)Atchison,R.W.,Casto,B.C.,and Hammond,W.M.(1965).Adenovirus-associated defective virus particles.Science 149,754–756.
(3)Buller,R.M.,Janik,J.E.,Sebring,E.D.,and Rose,J.A.(1981).Herpes simplex virus types 1and 2completely help adenovirusassociated virus replication.J.Virol.40,241–247.
(4)Berns,K.I.,and Linden,M.(1995).The cryptic life cycle of adenoassociated virus.Bio Essays 17,237–245.
(5)Hermonat,P.L.,Labow,M.A.,Wright,R.,Berns,K.I.,and Muzyczka,N.(1984).Genetics of adeno-associated virus:isolation and preliminary characterization of adeno-associated virus type 2mutants.J.Virol.51,329–339.
(6)Ni,T.H.,Zhou,X.,McCarty,D.M.,Zolotukhin,I.,and Muzyczka,N.(1994).In vitro replication of adeno-associated virus DNA.J.Virol.68,1128–1138.
(7)Richardson,W.D.,and Westphal,H.(1984).Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus.J.Virol.51,404–410.
(8)Samulski,R.J.,and Shenk,T.(1988).Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs.J.Virol.62,206–210.
(9)Jay,F.T.,Laughlin,C.A.,and Carter,B.J.(1981).Eukaryotic translational control:adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein.Proc.Natl.Acad.Sci.USA 78,2927–2931.
(10)Janik,J.E.,Huston,M.M.,Cho,K.,and Rose,J.A.(1989).Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA.Virology 168,320–329.
(11)Richardson,W.D.,and Westphal,H.(1981).A cascade of adenovirus early functions is required for expression of adeno-associated virus.Cell 27,133–141.
(12)Carter,B.J.,Marcus-Sekura,C.J.,Laughlin,C.A.,and Ketner,G.(1983).Properties of an adenovirus type 2mutant,Ad2dl807,having a deletion near the right-hand genome terminus:failure to help AAV replication.Virology 126,505–516.
(13)West,M.H.,Trempe,J.P.,Tratschin,J.D.,and Carter,B.J.(1987).Gene e
(14)xpression in adeno-associated virus vectors:the effects of chimeric mRNA structure,helper virus,and adenovirus VA1 RNA.Virology 160,38–47.
(15)Janik,J.E.,Huston,M.M.,Cho,K.,and Rose,J.A.(1989).Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA.Virology 168,320–329.
disclosure of Invention
The invention aims to optimize sequences on the basis of the pHelper (11K) plasmid adopted in the prior art and improve the efficiency of in vitro preparation of the adeno-associated virus.
In order to achieve the aim, the invention provides a recombinant plasmid based on an Adv5 sequence, which is used for in vitro preparation of adeno-associated virus and comprises a sequence derived from Adv5, wherein the sequence derived from Adv5 is shown as SEQ ID NO. 2.
Preferably, said plasmid further comprises a kanamycin resistance gene.
Preferably, the sequence of the plasmid is shown as SEQ ID NO. 1.
The present invention also provides an adeno-associated virus packaging system comprising: the recombinant plasmid based on the adenovirus type 5 sequence is described above.
Preferably, the system is transfected into 293 cells for adeno-associated virus production.
Compared with the prior art, the invention has the following beneficial effects:
the recombinant plasmid constructed based on the wild type 5 adenovirus (Adv) sequence expresses 5 adenovirus functional genes such as VA, E2A, E4 and the like after cells are transfected in vitro, can be used for in vitro adenovirus vector preparation, and has the virus packaging amount which is more than 5 times higher than that of the current 11K pHelper plasmid.
Drawings
FIG. 1 is a schematic diagram of a recombinant plasmid based on adenovirus type 5 (Adv5) sequence according to the present invention.
FIG. 2 is a schematic diagram showing the construction of the pHelper plasmid of the present invention.
FIG. 3 is a graph comparing the virus packaging efficiency of the pHelper (16K) plasmid of the present invention with that of the conventional pHelper (11K) plasmid.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
As shown in FIG. 1, the schematic diagram of the recombinant plasmid based on adenovirus type 5 (Adv5) sequence of the present invention is pHelper (16K) plasmid, and the sequence is shown in SEQ ID NO. 1. The invention carries out sequence optimization on the basis of the currently adopted pHelper (11K) plasmid, and Adv5 is used for replacing Adv2, starting from ColE1, and the nucleic acid position is 36-888 bp; the kanamycin resistance gene has the nucleic acid position of 903-1718 bp; the sequence derived from Adv5, the nucleic acid position is 2312-16105bp (shown in SEQ ID NO. 2). The recombinant plasmid of the invention, by cotransfection with pRC and pAAV-GOI plasmid in 293 cells, improves the packaging amount of virus by more than 5 times compared with the commonly used pHelper (11K) plasmid. The pRC plasmid encodes the rep and cap genes. The pAAV-GOI plasmid refers to a plasmid carrying a target gene, the plasmid has two inverted repeat sequences, and the target gene is carried between the two inverted repeat sequences. The plasmid of the invention can be applied to scientific research and large-scale preparation of adeno-associated virus, and greatly improves the production efficiency of adeno-associated virus.
FIG. 2 is a schematic diagram showing the construction of the pHelper (16K) plasmid of the present invention. As shown in FIG. 2, different sequences are synthesized by segmentation, and the different sequences are connected together by different endonucleases to form a complete plasmid. Different endonucleases are composed of different bases and can function to ligate different fragments. The endonuclease shown in FIG. 2 may be replaced by a different endonuclease, such as BamH1, Sal1, Xho1, Mfe1, etc. The specific construction process is as follows:
1) the pHelper (11K) ampicillin plasmid was changed to the kanamycin-resistant pHelper (11K, KanR) plasmid.
2) Three plasmids are synthesized by segmentation, and enzyme cutting sites are added at two ends of the three plasmids. Wherein, the first plasmid is added with BamH1 and Sal1 enzyme cutting sites. Xhol1 cleavage site was added to plasmid two. Plasmid three added mfe1 cleavage sites.
3) The plasmid was digested with BamH1 and Sal1 both overnight. The kanamycin-resistant pHelper (11K, KanR) plasmid was double digested with BamH1 and Sal1 overnight.
4) Gel recovery was performed using agarose gel DNA recovery kit.
5) The recovered fragments were ligated with T4 ligase.
6) Selecting single clone, carrying out single enzyme digestion by Xhol1, dephosphorylation, carrying out single enzyme digestion by Xhol1, carrying out gel recovery, and connecting the recovered fragments by T4 ligase.
7) Selecting single clone, carrying out single enzyme digestion by Mfe1, dephosphorylation, carrying out single enzyme digestion by Mfe1, carrying out gel recovery, and connecting the recovered fragment by T4 ligase.
8) Selecting single clone, enzyme digestion verification and sequencing to obtain correct plasmid.
FIG. 3 is a graph showing the comparison of the virus packaging efficiency of the pHelper (16K) plasmid of the present invention with that of the commonly used pHelper (11K) plasmid. The specific experimental process is as follows:
1) the viral vectors were obtained by the plasmid cotransfection method. 293 suspension cells were cultured to a density suitable for cell transfection, and a pRC plasmid containing AAV coat protein genes, a pAAV-GFP plasmid, and helper plasmids 11K and 16K for genes that facilitate AAV replication were co-transfected in HEK293 cells to initially form recombinant adeno-associated viruses.
2) Cells were harvested after 72h of transfection.
3) After cracking, MgCl2(Sigma 442611-500GM), NP9(Sigma NP9-500ML), and the like.
4) The affinity filler was equilibrated with Poros-AAVX affinity resin (Thermofeisher A36739) to adsorb AAV.
5) High salt washes were performed with 20mM Tris-Base (Sigma 648310-2.5 KG-M).
6) Low salt washes were performed with 1M NaCl (Sigma S7653), pH 7.5.
7) The above eluate was neutralized with 200mM Tris-Base (Sigma 648310-2.5KG-M), 0.01% Pluronic F68 (Thermofoisher 24040032).
8) And (4) performing centrifugal concentration and ultrafiltration, and determining the titer of the virus by a real-time fluorescent quantitative PCR method.
As can be seen from FIG. 3, the pHelper (16K) plasmid of the present invention has a higher viral packaging capacity and a 5-fold higher titer (titer) than the commonly used pHelper (11K) research plasmid.
In conclusion, the sequence of the conventional pHelper (11K) plasmid is optimized, Adv5 is used to replace Adv2, and the pHelper (16K) kana plasmid designed by the invention is subjected to cotransformation with pRC and pAAV-GFP three plasmids in 293 cells, so that the virus content of the pHelper (11K) helper plasmid virus package is higher than that of the conventional pHelper (11K) helper plasmid virus package by more than 5 times.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
<110> first-person hospital in Shanghai City
<120> recombinant plasmid based on type 5 adenovirus sequence and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16105
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtcgacgttt aaaccatatg atcagctcac tcaaaggcgg taatacggtt atccacagaa 60
tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 120
aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 180
aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 240
ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 300
tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 360
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 420
gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 480
tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 540
acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 600
tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 660
caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 720
aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 780
aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 840
ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 900
agttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 960
ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 1020
cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 1080
cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 1140
actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc 1200
cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 1260
tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 1320
gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 1380
tattcttcta atacctggaa tgctgttttc ccagggatcg cagtggtgag taaccatgca 1440
tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 1500
tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 1560
aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 1620
acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 1680
ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat 1740
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 1800
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg 1860
ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 1920
aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 1980
ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 2040
gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 2100
tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag 2160
cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 2220
gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 2280
ttaatgcgcc gctacagggc gcgatggatc ctcgacagaa gcaccatgtc cttgggtccg 2340
gcctgctgaa tgcgcaggcg gtcggccatg ccccaggctt cgttttgaca tcggcgcagg 2400
tctttgtagt agtcttgcat gagcctttct accggcactt cttcttctcc ttcctcttgt 2460
cctgcatctc ttgcatctat cgctgcggcg gcggcggagt ttggccgtag gtggcgccct 2520
cttcctccca tgcgtgtgac cccgaagccc ctcatcggct gaagcagggc taggtcggcg 2580
acaacgcgct cggctaatat ggcctgctgc acctgcgtga gggtagactg gaagtcatcc 2640
atgtccacaa agcggtggta tgcgcccgtg ttgatggtgt aagtgcagtt ggccataacg 2700
gaccagttaa cggtctggtg acccggctgc gagagctcgg tgtacctgag acgcgagtaa 2760
gccctcgagt caaatacgta gtcgttgcaa gtccgcacca ggtactggta tcccaccaaa 2820
aagtgcggcg gcggctggcg gtagaggggc cagcgtaggg tggccggggc tccgggggcg 2880
agatcttcca acataaggcg atgatatccg tagatgtacc tggacatcca ggtgatgccg 2940
gcggcggtgg tggaggcgcg cggaaagtcg cggacgcggt tccagatgtt gcgcagcggc 3000
aaaaagtgct ccatggtcgg gacgctctgg ccggtcaggc gcgcgcaatc gttgacgctc 3060
tagaccgtgc aaaaggagag cctgtaagcg ggcactcttc cgtggtctgg tggataaatt 3120
cgcaagggta tcatggcgga cgaccggggt tcgagccccg tatccggccg tccgccgtga 3180
tccatgcggt taccgcccgc gtgtcgaacc caggtgtgcg acgtcagaca acgggggagt 3240
gctccttttg gcttccttcc aggcgcggcg gctgctgcgc tagctttttt ggccactggc 3300
cgcgcgcagc gtaagcggtt aggctggaaa gcgaaagcat taagtggctc gctccctgta 3360
gccggagggt tattttccaa gggttgagtc gcgggacccc cggttcgagt ctcggaccgg 3420
ccggactgcg gcgaacgggg gtttgcctcc ccgtcatgca agaccccgct tgcaaattcc 3480
tccggaaaca gggacgagcc ccttttttgc ttttcccaga tgcatccggt gctgcggcag 3540
atgcgccccc ctcctcagca gcggcaagag caagagcagc ggcagacatg cagggcaccc 3600
tcccctcctc ctaccgcgtc aggaggggcg acatccgcgg ttgacgcggc agcagatggt 3660
gattacgaac ccccgcggcg ccgggcccgg cactacctgg acttggagga gggcgagggc 3720
ctggcgcggc taggagcgcc ctctcctgag cggtacccaa gggtgcagct gaagcgtgat 3780
acgcgtgagg cgtacgtgcc gcggcagaac ctgtttcgcg accgcgaggg agaggagccc 3840
gaggagatgc gggatcgaaa gttccacgca gggcgcgagc tgcggcatgg cctgaatcgc 3900
gagcggttgc tgcgcgagga ggactttgag cccgacgcgc gaaccgggat tagtcccgcg 3960
cgcgcacacg tggcggccgc cgacctggta accgcatacg agcagacggt gaaccaggag 4020
attaactttc aaaaaagctt taacaaccac gtgcgtacgc ttgtggcgcg cgaggaggtg 4080
gctataggac tgatgcatct gtgggacttt gtaagcgcgc tggagcaaaa cccaaatagc 4140
aagccgctca tggcgcagct gttccttata gtgcagcaca gcagggacaa cgaggcattc 4200
agggatgcgc tgctaaacat agtagagccc gagggccgct ggctgctcga tttgataaac 4260
atcctgcaga gcatagtggt gcaggagcgc agcttgagcc tggctgacaa ggtggccgcc 4320
atcaactatt ccatgcttag cctgggcaag ttttacgccc gcaagatata ccatacccct 4380
tacgttccca tagacaagga ggtaaagatc gaggggttct acatgcgcat ggcgctgaag 4440
gtgcttacct tgagcgacga cctgggcgtt tatcgcaacg agcgcatcca caaggccgtg 4500
agcgtgagcc ggcggcgcga gctcagcgac cgcgagctga tgcacagcct gcaaagggcc 4560
ctggctggca cgggcagcgg cgatagagag gccgagtcct actttgacgc gggcgctgac 4620
ctgcgctggg ccccaagccg acgcgccctg gaggcagctg gggccggacc tgggctggcg 4680
gtggcacccg cgcgcgctgg caacgtcggc ggcgtggagg aatatgacga ggacgatgag 4740
tacgagccag aggacggcga gtactaagcg gtgatgtttc tgatcagatg atgcaagacg 4800
caacggaccc ggcggtgcgg gcggcgctgc agagccagcc gtccggcctt aactccacgg 4860
acgactggcg ccaggtcatg gaccgcatca tgtcgctgac tgcgcgcaat cctgacgcgt 4920
tccggcagca gccgcaggcc aaccggctct ccgcaattct ggaagcggtg gtcccggcgc 4980
gcgcaaaccc cacgcacgag aaggtgctgg cgatcgtaaa cgcgctggcc gaaaacaggg 5040
ccatatgggc tccagtgagc aggaactgaa agccattgtc aaagatcttg gttgtgggcc 5100
atattttttg ggcacctatg acaagcgctt tccaggcttt gtttctccac acaagctcgc 5160
ctgcgccata gtcaatacgg ccggtcgcga gactgggggc gtacactgga tggcctttgc 5220
ctggaacccg cactcaaaaa catgctacct ctttgagccc tttggctttt ctgaccagcg 5280
actcaagcag gtttaccagt ttgagtacga gtcactcctg cgccgtagcg ccattgcttc 5340
ttcccccgac cgctgtataa cgctggaaaa gtccacccaa agcgtacagg ggcccaactc 5400
ggccgcctgt ggactattct gctgcatgtt tctccacgcc tttgccaact ggccccaaac 5460
tcccatggat cacaacccca ccatgaacct tattaccggg gtacccaact ccatgctcaa 5520
cagtccccag gtacagccca ccctgcgtcg caaccaggaa cagctctaca gcttcctgga 5580
gcgccactcg ccctacttcc gcagccacag tgcgcagatt aggagcgcca cttctttttg 5640
tcacttgaaa aacatgtaaa aataatgtac tagagacact ttcaataaag gcaaatgctt 5700
ttatttgtac actctcgggt gattatttac ccccaccctt gccgtctgcg ccgtttaaaa 5760
atcaaagggg ttctgccgcg catcgctatg cgccactggc agggacacgt tgcgatactg 5820
gtgtttagtg ctccacttaa actcaggcac aaccatccgc ggcagctcgg tgaagttttc 5880
actccacagg ctgcgcacca tcaccaacgc gtttagcagg tcgggcgccg atatcttgaa 5940
gtcgcagttg gggcctccgc cctgcgcgcg cgagttgcga tacacagggt tgcagcactg 6000
gaacactatc agcgccgggt ggtgcacgct ggccagcacg ctcttgtcgg agatcagatc 6060
cgcgtccagg tcctccgcgt tgctcagggc gaacggagtc aactttggta gctgccttcc 6120
caaaaagggc gcgtgcccag gctttgagtt gcactcgcac cgtagtggca tcaaaaggtg 6180
accgtgcccg gtctgggcgt taggatacag cgcctgcata aaagccttga tctgcttaaa 6240
agccacctga gcctttgcgc cttcagagaa gaacatgccg caagacttgc cggaaaactg 6300
attggccgga caggccgcgt cgtgcacgca gcaccttgcg tcggtgttgg agatctgcac 6360
cacatttcgg ccccaccggt tcttcacgat cttggccttg ctagactgct ccttcagcgc 6420
gcgctgcccg ttttcgctcg tcacatccat ttcaatcacg tgctccttat ttatcataat 6480
gcttccgtgt agacacttaa gctcgccttc gatctcagcg cagcggtgca gccacaacgc 6540
gcagcccgtg ggctcgtgat gcttgtaggt cacctctgca aacgactgca ggtacgcctg 6600
caggaatcgc cccatcatcg tcacaaaggt cttgttgctg gtgaaggtca gctgcaaccc 6660
gcggtgctcc tcgttcagcc aggtcttgca tacggccgcc agagcttcca cttggtcagg 6720
cagtagtttg aagttcgcct ttagatcgtt atccacgtgg tacttgtcca tcagcgcgcg 6780
cgcagcctcc atgcccttct cccacgcaga cacgatcggc acactcagcg ggttcatcac 6840
cgtaatttca ctttccgctt cgctgggctc ttcctcttcc tcttgcgtcc gcataccacg 6900
cgccactggg tcgtcttcat tcagccgccg cactgtgcgc ttacctcctt tgccatgctt 6960
gattagcacc ggtgggttgc tgaaacccac catttgtagc gccacatctt ctctttcttc 7020
ctcgctgtcc acgattacct ctggtgatgg cgggcgctcg ggcttgggag aagggcgctt 7080
ctttttcttc ttgggcgcaa tggccaaatc cgccgccgag gtcgatggcc gcgggctggg 7140
tgtgcgcggc accagcgcgt cttgtgatga gtcttcctcg tcctcggact cgatacgccg 7200
cctcatccgc ttttttgggg gcgcccgggg aggcggcggc gacggggacg gggacgacac 7260
gtcctccatg gttgggggac gtcgcgccgc accgcgtccg cgctcggggg tggtttcgcg 7320
ctgctcctct tcccgactgg ccatttcctt ctcctatagg cagaaaaaga tcatggagtc 7380
agtcgagaag aaggacagcc taaccgcccc ctctgagttc gccaccaccg cctccaccga 7440
tgccgccaac gcgcctacca ccttccccgt cgaggcaccc ccgcttgagg aggaggaagt 7500
gattatcgag caggacccag gttttgtaag cgaagacgac gaggaccgct cagtaccaac 7560
agaggataaa aagcaagacc aggacaacgc agaggcaaac gaggaacaag tcgggcgggg 7620
ggacgaaagg catggcgact acctagatgt gggagacgac gtgctgttga agcatctgca 7680
gcgccagtgc gccattatct gcgacgcgtt gcaagagcgc agcgatgtgc ccctcgccat 7740
agcggatgtc agccttgcct acgaacgcca cctattctca ccgcgcgtac cccccaaacg 7800
ccaagaaaac ggcacatgcg agcccaaccc gcgcctcaac ttctaccccg tatttgccgt 7860
gccagaggtg cttgccacct atcacatctt tttccaaaac tgcaagatac ccctatcctg 7920
ccgtgccaac cgcagccgag cggacaagca gctggccttg cggcagggcg ctgtcatacc 7980
tgatatcgcc tcgctcaacg aagtgccaaa aatctttgag ggtcttggac gcgacgagaa 8040
gcgcgcggca aacgctctgc aacaggaaaa cagcgaaaat gaaagtcact ctggagtgtt 8100
ggtggaactc gagggtgaca acgcgcgcct agccgtacta aaacgcagca tcgaggtcac 8160
ccactttgcc tacccggcac ttaacctacc ccccaaggtc atgagcacag tcatgagtga 8220
gctgatcgtg cgccgtgcgc agcccctgga gagggatgca aatttgcaag aacaaacaga 8280
ggagggccta cccgcagttg gcgacgagca gctagcgcgc tggcttcaaa cgcgcgagcc 8340
tgccgacttg gaggagcgac gcaaactaat gatggccgca gtgctcgtta ccgtggagct 8400
tgagtgcatg cagcggttct ttgctgaccc ggagatgcag cgcaagctag aggaaacatt 8460
gcactacacc tttcgacagg gctacgtacg ccaggcctgc aagatctcca acgtggagct 8520
ctgcaacctg gtctcctacc ttggaatttt gcacgaaaac cgccttgggc aaaacgtgct 8580
tcattccacg ctcaagggcg aggcgcgccg cgactacgtc cgcgactgcg tttacttatt 8640
tctatgctac acctggcaga cggccatggg cgtttggcag cagtgcttgg aggagtgcaa 8700
cctcaaggag ctgcagaaac tgctaaagca aaacttgaag gacctatgga cggccttcaa 8760
cgagcgctcc gtggccgcgc acctggcgga catcattttc cccgaacgcc tgcttaaaac 8820
cctgcaacag ggtctgccag acttcaccag tcaaagcatg ttgcagaact ttaggaactt 8880
tatcctagag cgctcaggaa tcttgcccgc cacctgctgt gcacttccta gcgactttgt 8940
gcccattaag taccgcgaat gccctccgcc gctttggggc cactgctacc ttctgcagct 9000
agccaactac cttgcctacc actctgacat aatggaagac gtgagcggtg acggtctact 9060
ggagtgtcac tgtcgctgca acctatgcac cccgcaccgc tccctggttt gcaattcgca 9120
gctgcttaac gaaagtcaaa ttatcggtac ctttgagctg cagggtccct cgcctgacga 9180
aaagtccgcg gctccggggt tgaaactcac tccggggctg tggacgtcgg cttaccttcg 9240
caaatttgta cctgaggact accacgccca cgagattagg ttctacgaag accaatcccg 9300
cccgccaaat gcggagctta ccgcctgcgt cattacccag ggccacattc ttggccaatt 9360
gcaagccatc aacaaagccc gccaagagtt tctgctacga aagggacggg gggtttactt 9420
ggacccccag tccggcgagg agctcaaccc aatccccccg ccgccgcagc cctatcagca 9480
gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa gaagctgcag ctgccgccgc 9540
cacccacgga cgaggaggaa tactgggaca gtcaggcaga ggaggttttg gacgaggagg 9600
aggaggacat gatggaagac tgggagagcc tagacgagga agcttccgag gtcgaagagg 9660
tgtcagacga aacaccgtca ccctcggtcg cattcccctc gccggcgccc cagaaatcgg 9720
caaccggttc cagcatggct acaacctccg ctcctcaggc gccgccggca ctgcccgttc 9780
gccgacccaa ccgtagatgg gacaccactg gaaccagggc cggtaagtcc aagcagccgc 9840
cgccgttagc ccaagagcaa caacagcgcc aaggctaccg ctcatggcgc gggcacaaga 9900
acgccatagt tgcttgcttg caagactgtg ggggcaacat ctccttcgcc cgccgctttc 9960
ttctctacca tcacggcgtg gccttccccc gtaacatcct gcattactac cgtcatctct 10020
acagcccata ctgcaccggc ggcagcggca gcggcagcaa cagcagcggc cacacagaag 10080
caaaggcgac cggatagcaa gactctgaca aagcccaaga aatccacagc ggcggcagca 10140
gcaggaggag gagcgctgcg tctggcgccc aacgaacccg tatcgacccg cgagcttaga 10200
aacaggattt ttcccactct gtatgctata tttcaacaga gcaggggcca agaacaagag 10260
ctgaaaataa aaaacaggtc tctgcgatcc ctcacccgca gctgcctgta tcacaaaagc 10320
gaagatcagc ttcggcgcac gctggaagac gcggaggctc tcttcagtaa atactgcgcg 10380
ctgactctta aggactagtt tcgcgccctt tctcaaattt aagcgcgaaa actacgtcat 10440
ctccagcggc cacacccggc gccagcacct gtcgtcagcg ccattatgag caaggaaatt 10500
cccacgccct acatgtggag ttaccagcca caaatgggac ttgcggctgg agctgcccaa 10560
gactactcaa cccgaataaa ctacatgagc gcgggacccc acatgatatc ccgggtcaac 10620
ggaatccgcg cccaccgaaa ccgaattctc ttggaacagg cggctattac caccacacct 10680
cgtaataacc ttaatccccg tagttggccc gctgccctgg tgtaccagga aagtcccgct 10740
cccaccactg tggtacttcc cagagacgcc caggccgaag ttcagatgac taactcaggg 10800
gcgcagcttg cgggcggctt tcgtcacagg gtgcggtcgc ccgggcaggg tataactcac 10860
ctgacaatca gagggcgagg tattcagctc aacgacgagt cggtgagctc ctcgcttggt 10920
ctccgtccgg acgggacatt tcagatcggc ggcgccggcc gtccttcatt cacgcctcgt 10980
caggcaatcc taactctgca gacctcgtcc tctgagccgc gctctggagg cattggaact 11040
ctgcaattta ttgaggagtt tgtgccatcg gtctacttta accccttctc gggacctccc 11100
ggccactatc cggatcaatt tattcctaac tttgacgcgg taaaggactc ggcggacggc 11160
tacgactgaa tgtcagtttc ctcctgttcc tgtccatccg cacccactat cttcatgttg 11220
ttgcagatga agcgcgcaag accgtctgaa gataccttca accccgtgta tccatatgac 11280
acggaaaccg gtcctccaac tgtgcctttt cttactcctc cctttgtatc ccccaatggg 11340
tttcaagaga gtccccctgg ggtactctct ttgcgcctat ccgaacctct agttacctcc 11400
aatggcatgc ttgcgctcaa aatgggcaac ggcctctctc tggacgaggc cggcaacctt 11460
acctcccaaa atgtaaccac tgtgagccca cctctcaaaa aaaccaagtc aaacataaac 11520
ctggaaatat ctgcacccct cacagttacc tcagaagccc taactgtggc tgccgccgca 11580
cctctaatgg tcgcgggcaa cacactcacc atgcaatcac aggccccgct aaccgtgcac 11640
gactccaaac ttagcattgc cacccaagga cccctcacag tgtcagaagg aaagctagcc 11700
ctgcaaacat caggccccct caccaccacc gatagcagta cccttactat cactgcctca 11760
ccccctctaa ctactgccac tggtagcttg ggcattgact tgaaagagcc catttataca 11820
caaaatggaa aactaggact aaagtacggg gctcctttgc atgtaacaga cgacctaaac 11880
actttgaccg tagcaactgg tccaggtgtg actattaata atacttcctt gcaaactaaa 11940
gttactggag ccttgggttt tgattcacaa ggcaatatgc aacttaatgt agcaggagga 12000
ctaaggattg attctcaaaa cagacgcctt atacttgatg ttagttatcc gtttgatgct 12060
caaaaccaac taaatctaag actaggacag ggccctcttt ttataaactc agcccacaac 12120
ttggatatta actacaacaa aggcctttac ttgtttacag cttcaaacaa ttccaaaaag 12180
cttgaggtta acctaagcac tgccaagggg ttgatgtttg acgctacagc catagccatt 12240
aatgcaggag atgggcttga atttggttca cctaatgcac caaacacaaa tcccctcaaa 12300
acaaaaattg gccatggcct agaatttgat tcaaacaagg ctatggttcc taaactagga 12360
actggcctta gttttgacag cacaggtgcc attacagtag gaaacaaaaa taatgataag 12420
ctaactttgt ggaccacacc agctccatct cctaactgta gactaaatgc agagaaagat 12480
gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa tacttgctac agtttcagtt 12540
ttggctgtta aaggcagttt ggctccaata tctggaacag ttcaaagtgc tcatcttatt 12600
ataagatttg acgaaaatgg agtgctacta aacaattcct tcctggaccc agaatattgg 12660
aactttagaa atggagatct tactgaaggc acagcctata caaacgctgt tggatttatg 12720
cctaacctat cagcttatcc aaaatctcac ggtaaaactg ccaaaagtaa cattgtcagt 12780
caagtttact taaacggaga caaaactaaa cctgtaacac taaccattac actaaacggt 12840
acacaggaaa caggagacac aactccaagt gcatactcta tgtcattttc atgggactgg 12900
tctggccaca actacattaa tgaaatattt gccacatcct cttacacttt ttcatacatt 12960
gcccaagaat aaagaatcgt ttgtgttatg tttcaacgtg tttatttttc aattgcagaa 13020
aatttcaagt catttttcat tcagtagtat agccccacca ccacatagct tatacagatc 13080
accgtacctt aatcaaactc acagaaccct agtattcaac ctgccacctc cctcccaaca 13140
cacagagtac acagtccttt ctccccggct ggccttaaaa agcatcatat catgggtaac 13200
agacatattc ttaggtgtta tattccacac ggtttcctgt cgagccaaac gctcatcagt 13260
gatattaata aactccccgg gcagctcact taagttcatg tcgctgtcca gctgctgagc 13320
cacaggctgc tgtccaactt gcggttgctt aacgggcggc gaaggagaag tccacgccta 13380
catgggggta gagtcataat cgtgcatcag gatagggcgg tggtgctgca gcagcgcgcg 13440
aataaactgc tgccgccgcc gctccgtcct gcaggaatac aacatggcag tggtctcctc 13500
agcgatgatt cgcaccgccc gcagcataag gcgccttgtc ctccgggcac agcagcgcac 13560
cctgatctca cttaaatcag cacagtaact gcagcacagc accacaatat tgttcaaaat 13620
cccacagtgc aaggcgctgt atccaaagct catggcgggg accacagaac ccacgtggcc 13680
atcataccac aagcgcaggt agattaagtg gcgacccctc ataaacacgc tggacataaa 13740
cattacctct tttggcatgt tgtaattcac cacctcccgg taccatataa acctctgatt 13800
aaacatggcg ccatccacca ccatcctaaa ccagctggcc aaaacctgcc cgccggctat 13860
acactgcagg gaaccgggac tggaacaatg acagtggaga gcccaggact cgtaaccatg 13920
gatcatcatg ctcgtcatga tatcaatgtt ggcacaacac aggcacacgt gcatacactt 13980
cctcaggatt acaagctcct cccgcgttag aaccatatcc cagggaacaa cccattcctg 14040
aatcagcgta aatcccacac tgcagggaag acctcgcacg taactcacgt tgtgcattgt 14100
caaagtgtta cattcgggca gcagcggatg atcctccagt atggtagcgc gggtttctgt 14160
ctcaaaagga ggtagacgat ccctactgta cggagtgcgc cgagacaacc gagatcgtgt 14220
tggtcgtagt gtcatgccaa atggaacgcc ggacgtagtc atatttcctg aagcaaaacc 14280
aggtgcgggc gtgacaaaca gatctgcgtc tccggtctcg ccgcttagat cgctctgtgt 14340
agtagttgta gtatatccac tctctcaaag catccaggcg ccccctggct tcgggttcta 14400
tgtaaactcc ttcatgcgcc gctgccctga taacatccac caccgcagaa taagccacac 14460
ccagccaacc tacacattcg ttctgcgagt cacacacggg aggagcggga agagctggaa 14520
gaaccatgtt ttttttttta ttccaaaaga ttatccaaaa cctcaaaatg aagatctatt 14580
aagtgaacgc gctcccctcc ggtggcgtgg tcaaactcta cagccaaaga acagataatg 14640
gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa cggccctcac gtccaagtgg 14700
acgtaaaggc taaacccttc agggtgaatc tcctctataa acattccagc accttcaacc 14760
atgcccaaat aattctcatc tcgccacctt ctcaatatat ctctaagcaa atcccgaata 14820
ttaagtccgg ccattgtaaa aatctgctcc agagcgccct ccaccttcag cctcaagcag 14880
cgaatcatga ttgcaaaaat tcaggttcct cacagacctg tataagattc aaaagcggaa 14940
cattaacaaa aataccgcga tcccgtaggt cccttcgcag ggccagctga acataatcgt 15000
gcaggtctgc acggaccagc gcggccactt ccccgccagg aaccatgaca aaagaaccca 15060
cactgattat gacacgcata ctcggagcta tgctaaccag cgtagccccg atgtaagctt 15120
gttgcatggg cggcgatata aaatgcaagg tgctgctcaa aaaatcaggc aaagcctcgc 15180
gcaaaaaaga aagcacatcg tagtcatgct catgcagata aaggcaggta agctccggaa 15240
ccaccacaga aaaagacacc atttttctct caaacatgtc tgcgggtttc tgcataaaca 15300
caaaataaaa taacaaaaaa acatttaaac attagaagcc tgtcttacaa caggaaaaac 15360
aacccttata agcataagac ggactacggc catgccggcg tgaccgtaaa aaaactggtc 15420
accgtgatta aaaagcacca ccgacagctc ctcggtcatg tccggagtca taatgtaaga 15480
ctcggtaaac acatcaggtt gattcacatc ggtcagtgct aaaaagcgac cgaaatagcc 15540
cgggggaata catacccgca ggcgtagaga caacattaca gcccccatag gaggtataac 15600
aaaattaata ggagagaaaa acacataaac acctgaaaaa ccctcctgcc taggcaaaat 15660
agcaccctcc cgctccagaa caacatacag cgcttccaca gcggcagcca taacagtcag 15720
ccttaccagt aaaaaagaaa acctattaaa aaaacaccac tcgacacggc accagctcaa 15780
tcagtcacag tgtaaaaaag ggccaagtgc agagcgagta tatataggac taaaaaatga 15840
cgtaacggtt aaagtccaca aaaaacaccc agaaaaccgc acgcgaacct acgcccagaa 15900
acgaaagcca aaaaacccac aacttcctca aatcgtcact tccgttttcc cacgttacgt 15960
aacttcccat tttaagaaaa ctacaattcc caacacatac aagttactcc gccctaaaac 16020
ctacgtcacc cgccccgttc ccacgccccg cgccacgtca caaactccac cccctcatta 16080
tcatattggc ttcaatccaa aataa 16105
<210> 2
<211> 13794
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcgacagaag caccatgtcc ttgggtccgg cctgctgaat gcgcaggcgg tcggccatgc 60
cccaggcttc gttttgacat cggcgcaggt ctttgtagta gtcttgcatg agcctttcta 120
ccggcacttc ttcttctcct tcctcttgtc ctgcatctct tgcatctatc gctgcggcgg 180
cggcggagtt tggccgtagg tggcgccctc ttcctcccat gcgtgtgacc ccgaagcccc 240
tcatcggctg aagcagggct aggtcggcga caacgcgctc ggctaatatg gcctgctgca 300
cctgcgtgag ggtagactgg aagtcatcca tgtccacaaa gcggtggtat gcgcccgtgt 360
tgatggtgta agtgcagttg gccataacgg accagttaac ggtctggtga cccggctgcg 420
agagctcggt gtacctgaga cgcgagtaag ccctcgagtc aaatacgtag tcgttgcaag 480
tccgcaccag gtactggtat cccaccaaaa agtgcggcgg cggctggcgg tagaggggcc 540
agcgtagggt ggccggggct ccgggggcga gatcttccaa cataaggcga tgatatccgt 600
agatgtacct ggacatccag gtgatgccgg cggcggtggt ggaggcgcgc ggaaagtcgc 660
ggacgcggtt ccagatgttg cgcagcggca aaaagtgctc catggtcggg acgctctggc 720
cggtcaggcg cgcgcaatcg ttgacgctct agaccgtgca aaaggagagc ctgtaagcgg 780
gcactcttcc gtggtctggt ggataaattc gcaagggtat catggcggac gaccggggtt 840
cgagccccgt atccggccgt ccgccgtgat ccatgcggtt accgcccgcg tgtcgaaccc 900
aggtgtgcga cgtcagacaa cgggggagtg ctccttttgg cttccttcca ggcgcggcgg 960
ctgctgcgct agcttttttg gccactggcc gcgcgcagcg taagcggtta ggctggaaag 1020
cgaaagcatt aagtggctcg ctccctgtag ccggagggtt attttccaag ggttgagtcg 1080
cgggaccccc ggttcgagtc tcggaccggc cggactgcgg cgaacggggg tttgcctccc 1140
cgtcatgcaa gaccccgctt gcaaattcct ccggaaacag ggacgagccc cttttttgct 1200
tttcccagat gcatccggtg ctgcggcaga tgcgcccccc tcctcagcag cggcaagagc 1260
aagagcagcg gcagacatgc agggcaccct cccctcctcc taccgcgtca ggaggggcga 1320
catccgcggt tgacgcggca gcagatggtg attacgaacc cccgcggcgc cgggcccggc 1380
actacctgga cttggaggag ggcgagggcc tggcgcggct aggagcgccc tctcctgagc 1440
ggtacccaag ggtgcagctg aagcgtgata cgcgtgaggc gtacgtgccg cggcagaacc 1500
tgtttcgcga ccgcgaggga gaggagcccg aggagatgcg ggatcgaaag ttccacgcag 1560
ggcgcgagct gcggcatggc ctgaatcgcg agcggttgct gcgcgaggag gactttgagc 1620
ccgacgcgcg aaccgggatt agtcccgcgc gcgcacacgt ggcggccgcc gacctggtaa 1680
ccgcatacga gcagacggtg aaccaggaga ttaactttca aaaaagcttt aacaaccacg 1740
tgcgtacgct tgtggcgcgc gaggaggtgg ctataggact gatgcatctg tgggactttg 1800
taagcgcgct ggagcaaaac ccaaatagca agccgctcat ggcgcagctg ttccttatag 1860
tgcagcacag cagggacaac gaggcattca gggatgcgct gctaaacata gtagagcccg 1920
agggccgctg gctgctcgat ttgataaaca tcctgcagag catagtggtg caggagcgca 1980
gcttgagcct ggctgacaag gtggccgcca tcaactattc catgcttagc ctgggcaagt 2040
tttacgcccg caagatatac catacccctt acgttcccat agacaaggag gtaaagatcg 2100
aggggttcta catgcgcatg gcgctgaagg tgcttacctt gagcgacgac ctgggcgttt 2160
atcgcaacga gcgcatccac aaggccgtga gcgtgagccg gcggcgcgag ctcagcgacc 2220
gcgagctgat gcacagcctg caaagggccc tggctggcac gggcagcggc gatagagagg 2280
ccgagtccta ctttgacgcg ggcgctgacc tgcgctgggc cccaagccga cgcgccctgg 2340
aggcagctgg ggccggacct gggctggcgg tggcacccgc gcgcgctggc aacgtcggcg 2400
gcgtggagga atatgacgag gacgatgagt acgagccaga ggacggcgag tactaagcgg 2460
tgatgtttct gatcagatga tgcaagacgc aacggacccg gcggtgcggg cggcgctgca 2520
gagccagccg tccggcctta actccacgga cgactggcgc caggtcatgg accgcatcat 2580
gtcgctgact gcgcgcaatc ctgacgcgtt ccggcagcag ccgcaggcca accggctctc 2640
cgcaattctg gaagcggtgg tcccggcgcg cgcaaacccc acgcacgaga aggtgctggc 2700
gatcgtaaac gcgctggccg aaaacagggc catatgggct ccagtgagca ggaactgaaa 2760
gccattgtca aagatcttgg ttgtgggcca tattttttgg gcacctatga caagcgcttt 2820
ccaggctttg tttctccaca caagctcgcc tgcgccatag tcaatacggc cggtcgcgag 2880
actgggggcg tacactggat ggcctttgcc tggaacccgc actcaaaaac atgctacctc 2940
tttgagccct ttggcttttc tgaccagcga ctcaagcagg tttaccagtt tgagtacgag 3000
tcactcctgc gccgtagcgc cattgcttct tcccccgacc gctgtataac gctggaaaag 3060
tccacccaaa gcgtacaggg gcccaactcg gccgcctgtg gactattctg ctgcatgttt 3120
ctccacgcct ttgccaactg gccccaaact cccatggatc acaaccccac catgaacctt 3180
attaccgggg tacccaactc catgctcaac agtccccagg tacagcccac cctgcgtcgc 3240
aaccaggaac agctctacag cttcctggag cgccactcgc cctacttccg cagccacagt 3300
gcgcagatta ggagcgccac ttctttttgt cacttgaaaa acatgtaaaa ataatgtact 3360
agagacactt tcaataaagg caaatgcttt tatttgtaca ctctcgggtg attatttacc 3420
cccacccttg ccgtctgcgc cgtttaaaaa tcaaaggggt tctgccgcgc atcgctatgc 3480
gccactggca gggacacgtt gcgatactgg tgtttagtgc tccacttaaa ctcaggcaca 3540
accatccgcg gcagctcggt gaagttttca ctccacaggc tgcgcaccat caccaacgcg 3600
tttagcaggt cgggcgccga tatcttgaag tcgcagttgg ggcctccgcc ctgcgcgcgc 3660
gagttgcgat acacagggtt gcagcactgg aacactatca gcgccgggtg gtgcacgctg 3720
gccagcacgc tcttgtcgga gatcagatcc gcgtccaggt cctccgcgtt gctcagggcg 3780
aacggagtca actttggtag ctgccttccc aaaaagggcg cgtgcccagg ctttgagttg 3840
cactcgcacc gtagtggcat caaaaggtga ccgtgcccgg tctgggcgtt aggatacagc 3900
gcctgcataa aagccttgat ctgcttaaaa gccacctgag cctttgcgcc ttcagagaag 3960
aacatgccgc aagacttgcc ggaaaactga ttggccggac aggccgcgtc gtgcacgcag 4020
caccttgcgt cggtgttgga gatctgcacc acatttcggc cccaccggtt cttcacgatc 4080
ttggccttgc tagactgctc cttcagcgcg cgctgcccgt tttcgctcgt cacatccatt 4140
tcaatcacgt gctccttatt tatcataatg cttccgtgta gacacttaag ctcgccttcg 4200
atctcagcgc agcggtgcag ccacaacgcg cagcccgtgg gctcgtgatg cttgtaggtc 4260
acctctgcaa acgactgcag gtacgcctgc aggaatcgcc ccatcatcgt cacaaaggtc 4320
ttgttgctgg tgaaggtcag ctgcaacccg cggtgctcct cgttcagcca ggtcttgcat 4380
acggccgcca gagcttccac ttggtcaggc agtagtttga agttcgcctt tagatcgtta 4440
tccacgtggt acttgtccat cagcgcgcgc gcagcctcca tgcccttctc ccacgcagac 4500
acgatcggca cactcagcgg gttcatcacc gtaatttcac tttccgcttc gctgggctct 4560
tcctcttcct cttgcgtccg cataccacgc gccactgggt cgtcttcatt cagccgccgc 4620
actgtgcgct tacctccttt gccatgcttg attagcaccg gtgggttgct gaaacccacc 4680
atttgtagcg ccacatcttc tctttcttcc tcgctgtcca cgattacctc tggtgatggc 4740
gggcgctcgg gcttgggaga agggcgcttc tttttcttct tgggcgcaat ggccaaatcc 4800
gccgccgagg tcgatggccg cgggctgggt gtgcgcggca ccagcgcgtc ttgtgatgag 4860
tcttcctcgt cctcggactc gatacgccgc ctcatccgct tttttggggg cgcccgggga 4920
ggcggcggcg acggggacgg ggacgacacg tcctccatgg ttgggggacg tcgcgccgca 4980
ccgcgtccgc gctcgggggt ggtttcgcgc tgctcctctt cccgactggc catttccttc 5040
tcctataggc agaaaaagat catggagtca gtcgagaaga aggacagcct aaccgccccc 5100
tctgagttcg ccaccaccgc ctccaccgat gccgccaacg cgcctaccac cttccccgtc 5160
gaggcacccc cgcttgagga ggaggaagtg attatcgagc aggacccagg ttttgtaagc 5220
gaagacgacg aggaccgctc agtaccaaca gaggataaaa agcaagacca ggacaacgca 5280
gaggcaaacg aggaacaagt cgggcggggg gacgaaaggc atggcgacta cctagatgtg 5340
ggagacgacg tgctgttgaa gcatctgcag cgccagtgcg ccattatctg cgacgcgttg 5400
caagagcgca gcgatgtgcc cctcgccata gcggatgtca gccttgccta cgaacgccac 5460
ctattctcac cgcgcgtacc ccccaaacgc caagaaaacg gcacatgcga gcccaacccg 5520
cgcctcaact tctaccccgt atttgccgtg ccagaggtgc ttgccaccta tcacatcttt 5580
ttccaaaact gcaagatacc cctatcctgc cgtgccaacc gcagccgagc ggacaagcag 5640
ctggccttgc ggcagggcgc tgtcatacct gatatcgcct cgctcaacga agtgccaaaa 5700
atctttgagg gtcttggacg cgacgagaag cgcgcggcaa acgctctgca acaggaaaac 5760
agcgaaaatg aaagtcactc tggagtgttg gtggaactcg agggtgacaa cgcgcgccta 5820
gccgtactaa aacgcagcat cgaggtcacc cactttgcct acccggcact taacctaccc 5880
cccaaggtca tgagcacagt catgagtgag ctgatcgtgc gccgtgcgca gcccctggag 5940
agggatgcaa atttgcaaga acaaacagag gagggcctac ccgcagttgg cgacgagcag 6000
ctagcgcgct ggcttcaaac gcgcgagcct gccgacttgg aggagcgacg caaactaatg 6060
atggccgcag tgctcgttac cgtggagctt gagtgcatgc agcggttctt tgctgacccg 6120
gagatgcagc gcaagctaga ggaaacattg cactacacct ttcgacaggg ctacgtacgc 6180
caggcctgca agatctccaa cgtggagctc tgcaacctgg tctcctacct tggaattttg 6240
cacgaaaacc gccttgggca aaacgtgctt cattccacgc tcaagggcga ggcgcgccgc 6300
gactacgtcc gcgactgcgt ttacttattt ctatgctaca cctggcagac ggccatgggc 6360
gtttggcagc agtgcttgga ggagtgcaac ctcaaggagc tgcagaaact gctaaagcaa 6420
aacttgaagg acctatggac ggccttcaac gagcgctccg tggccgcgca cctggcggac 6480
atcattttcc ccgaacgcct gcttaaaacc ctgcaacagg gtctgccaga cttcaccagt 6540
caaagcatgt tgcagaactt taggaacttt atcctagagc gctcaggaat cttgcccgcc 6600
acctgctgtg cacttcctag cgactttgtg cccattaagt accgcgaatg ccctccgccg 6660
ctttggggcc actgctacct tctgcagcta gccaactacc ttgcctacca ctctgacata 6720
atggaagacg tgagcggtga cggtctactg gagtgtcact gtcgctgcaa cctatgcacc 6780
ccgcaccgct ccctggtttg caattcgcag ctgcttaacg aaagtcaaat tatcggtacc 6840
tttgagctgc agggtccctc gcctgacgaa aagtccgcgg ctccggggtt gaaactcact 6900
ccggggctgt ggacgtcggc ttaccttcgc aaatttgtac ctgaggacta ccacgcccac 6960
gagattaggt tctacgaaga ccaatcccgc ccgccaaatg cggagcttac cgcctgcgtc 7020
attacccagg gccacattct tggccaattg caagccatca acaaagcccg ccaagagttt 7080
ctgctacgaa agggacgggg ggtttacttg gacccccagt ccggcgagga gctcaaccca 7140
atccccccgc cgccgcagcc ctatcagcag cagccgcggg cccttgcttc ccaggatggc 7200
acccaaaaag aagctgcagc tgccgccgcc acccacggac gaggaggaat actgggacag 7260
tcaggcagag gaggttttgg acgaggagga ggaggacatg atggaagact gggagagcct 7320
agacgaggaa gcttccgagg tcgaagaggt gtcagacgaa acaccgtcac cctcggtcgc 7380
attcccctcg ccggcgcccc agaaatcggc aaccggttcc agcatggcta caacctccgc 7440
tcctcaggcg ccgccggcac tgcccgttcg ccgacccaac cgtagatggg acaccactgg 7500
aaccagggcc ggtaagtcca agcagccgcc gccgttagcc caagagcaac aacagcgcca 7560
aggctaccgc tcatggcgcg ggcacaagaa cgccatagtt gcttgcttgc aagactgtgg 7620
gggcaacatc tccttcgccc gccgctttct tctctaccat cacggcgtgg ccttcccccg 7680
taacatcctg cattactacc gtcatctcta cagcccatac tgcaccggcg gcagcggcag 7740
cggcagcaac agcagcggcc acacagaagc aaaggcgacc ggatagcaag actctgacaa 7800
agcccaagaa atccacagcg gcggcagcag caggaggagg agcgctgcgt ctggcgccca 7860
acgaacccgt atcgacccgc gagcttagaa acaggatttt tcccactctg tatgctatat 7920
ttcaacagag caggggccaa gaacaagagc tgaaaataaa aaacaggtct ctgcgatccc 7980
tcacccgcag ctgcctgtat cacaaaagcg aagatcagct tcggcgcacg ctggaagacg 8040
cggaggctct cttcagtaaa tactgcgcgc tgactcttaa ggactagttt cgcgcccttt 8100
ctcaaattta agcgcgaaaa ctacgtcatc tccagcggcc acacccggcg ccagcacctg 8160
tcgtcagcgc cattatgagc aaggaaattc ccacgcccta catgtggagt taccagccac 8220
aaatgggact tgcggctgga gctgcccaag actactcaac ccgaataaac tacatgagcg 8280
cgggacccca catgatatcc cgggtcaacg gaatccgcgc ccaccgaaac cgaattctct 8340
tggaacaggc ggctattacc accacacctc gtaataacct taatccccgt agttggcccg 8400
ctgccctggt gtaccaggaa agtcccgctc ccaccactgt ggtacttccc agagacgccc 8460
aggccgaagt tcagatgact aactcagggg cgcagcttgc gggcggcttt cgtcacaggg 8520
tgcggtcgcc cgggcagggt ataactcacc tgacaatcag agggcgaggt attcagctca 8580
acgacgagtc ggtgagctcc tcgcttggtc tccgtccgga cgggacattt cagatcggcg 8640
gcgccggccg tccttcattc acgcctcgtc aggcaatcct aactctgcag acctcgtcct 8700
ctgagccgcg ctctggaggc attggaactc tgcaatttat tgaggagttt gtgccatcgg 8760
tctactttaa ccccttctcg ggacctcccg gccactatcc ggatcaattt attcctaact 8820
ttgacgcggt aaaggactcg gcggacggct acgactgaat gtcagtttcc tcctgttcct 8880
gtccatccgc acccactatc ttcatgttgt tgcagatgaa gcgcgcaaga ccgtctgaag 8940
ataccttcaa ccccgtgtat ccatatgaca cggaaaccgg tcctccaact gtgccttttc 9000
ttactcctcc ctttgtatcc cccaatgggt ttcaagagag tccccctggg gtactctctt 9060
tgcgcctatc cgaacctcta gttacctcca atggcatgct tgcgctcaaa atgggcaacg 9120
gcctctctct ggacgaggcc ggcaacctta cctcccaaaa tgtaaccact gtgagcccac 9180
ctctcaaaaa aaccaagtca aacataaacc tggaaatatc tgcacccctc acagttacct 9240
cagaagccct aactgtggct gccgccgcac ctctaatggt cgcgggcaac acactcacca 9300
tgcaatcaca ggccccgcta accgtgcacg actccaaact tagcattgcc acccaaggac 9360
ccctcacagt gtcagaagga aagctagccc tgcaaacatc aggccccctc accaccaccg 9420
atagcagtac ccttactatc actgcctcac cccctctaac tactgccact ggtagcttgg 9480
gcattgactt gaaagagccc atttatacac aaaatggaaa actaggacta aagtacgggg 9540
ctcctttgca tgtaacagac gacctaaaca ctttgaccgt agcaactggt ccaggtgtga 9600
ctattaataa tacttccttg caaactaaag ttactggagc cttgggtttt gattcacaag 9660
gcaatatgca acttaatgta gcaggaggac taaggattga ttctcaaaac agacgcctta 9720
tacttgatgt tagttatccg tttgatgctc aaaaccaact aaatctaaga ctaggacagg 9780
gccctctttt tataaactca gcccacaact tggatattaa ctacaacaaa ggcctttact 9840
tgtttacagc ttcaaacaat tccaaaaagc ttgaggttaa cctaagcact gccaaggggt 9900
tgatgtttga cgctacagcc atagccatta atgcaggaga tgggcttgaa tttggttcac 9960
ctaatgcacc aaacacaaat cccctcaaaa caaaaattgg ccatggccta gaatttgatt 10020
caaacaaggc tatggttcct aaactaggaa ctggccttag ttttgacagc acaggtgcca 10080
ttacagtagg aaacaaaaat aatgataagc taactttgtg gaccacacca gctccatctc 10140
ctaactgtag actaaatgca gagaaagatg ctaaactcac tttggtctta acaaaatgtg 10200
gcagtcaaat acttgctaca gtttcagttt tggctgttaa aggcagtttg gctccaatat 10260
ctggaacagt tcaaagtgct catcttatta taagatttga cgaaaatgga gtgctactaa 10320
acaattcctt cctggaccca gaatattgga actttagaaa tggagatctt actgaaggca 10380
cagcctatac aaacgctgtt ggatttatgc ctaacctatc agcttatcca aaatctcacg 10440
gtaaaactgc caaaagtaac attgtcagtc aagtttactt aaacggagac aaaactaaac 10500
ctgtaacact aaccattaca ctaaacggta cacaggaaac aggagacaca actccaagtg 10560
catactctat gtcattttca tgggactggt ctggccacaa ctacattaat gaaatatttg 10620
ccacatcctc ttacactttt tcatacattg cccaagaata aagaatcgtt tgtgttatgt 10680
ttcaacgtgt ttatttttca attgcagaaa atttcaagtc atttttcatt cagtagtata 10740
gccccaccac cacatagctt atacagatca ccgtacctta atcaaactca cagaacccta 10800
gtattcaacc tgccacctcc ctcccaacac acagagtaca cagtcctttc tccccggctg 10860
gccttaaaaa gcatcatatc atgggtaaca gacatattct taggtgttat attccacacg 10920
gtttcctgtc gagccaaacg ctcatcagtg atattaataa actccccggg cagctcactt 10980
aagttcatgt cgctgtccag ctgctgagcc acaggctgct gtccaacttg cggttgctta 11040
acgggcggcg aaggagaagt ccacgcctac atgggggtag agtcataatc gtgcatcagg 11100
atagggcggt ggtgctgcag cagcgcgcga ataaactgct gccgccgccg ctccgtcctg 11160
caggaataca acatggcagt ggtctcctca gcgatgattc gcaccgcccg cagcataagg 11220
cgccttgtcc tccgggcaca gcagcgcacc ctgatctcac ttaaatcagc acagtaactg 11280
cagcacagca ccacaatatt gttcaaaatc ccacagtgca aggcgctgta tccaaagctc 11340
atggcgggga ccacagaacc cacgtggcca tcataccaca agcgcaggta gattaagtgg 11400
cgacccctca taaacacgct ggacataaac attacctctt ttggcatgtt gtaattcacc 11460
acctcccggt accatataaa cctctgatta aacatggcgc catccaccac catcctaaac 11520
cagctggcca aaacctgccc gccggctata cactgcaggg aaccgggact ggaacaatga 11580
cagtggagag cccaggactc gtaaccatgg atcatcatgc tcgtcatgat atcaatgttg 11640
gcacaacaca ggcacacgtg catacacttc ctcaggatta caagctcctc ccgcgttaga 11700
accatatccc agggaacaac ccattcctga atcagcgtaa atcccacact gcagggaaga 11760
cctcgcacgt aactcacgtt gtgcattgtc aaagtgttac attcgggcag cagcggatga 11820
tcctccagta tggtagcgcg ggtttctgtc tcaaaaggag gtagacgatc cctactgtac 11880
ggagtgcgcc gagacaaccg agatcgtgtt ggtcgtagtg tcatgccaaa tggaacgccg 11940
gacgtagtca tatttcctga agcaaaacca ggtgcgggcg tgacaaacag atctgcgtct 12000
ccggtctcgc cgcttagatc gctctgtgta gtagttgtag tatatccact ctctcaaagc 12060
atccaggcgc cccctggctt cgggttctat gtaaactcct tcatgcgccg ctgccctgat 12120
aacatccacc accgcagaat aagccacacc cagccaacct acacattcgt tctgcgagtc 12180
acacacggga ggagcgggaa gagctggaag aaccatgttt ttttttttat tccaaaagat 12240
tatccaaaac ctcaaaatga agatctatta agtgaacgcg ctcccctccg gtggcgtggt 12300
caaactctac agccaaagaa cagataatgg catttgtaag atgttgcaca atggcttcca 12360
aaaggcaaac ggccctcacg tccaagtgga cgtaaaggct aaacccttca gggtgaatct 12420
cctctataaa cattccagca ccttcaacca tgcccaaata attctcatct cgccaccttc 12480
tcaatatatc tctaagcaaa tcccgaatat taagtccggc cattgtaaaa atctgctcca 12540
gagcgccctc caccttcagc ctcaagcagc gaatcatgat tgcaaaaatt caggttcctc 12600
acagacctgt ataagattca aaagcggaac attaacaaaa ataccgcgat cccgtaggtc 12660
ccttcgcagg gccagctgaa cataatcgtg caggtctgca cggaccagcg cggccacttc 12720
cccgccagga accatgacaa aagaacccac actgattatg acacgcatac tcggagctat 12780
gctaaccagc gtagccccga tgtaagcttg ttgcatgggc ggcgatataa aatgcaaggt 12840
gctgctcaaa aaatcaggca aagcctcgcg caaaaaagaa agcacatcgt agtcatgctc 12900
atgcagataa aggcaggtaa gctccggaac caccacagaa aaagacacca tttttctctc 12960
aaacatgtct gcgggtttct gcataaacac aaaataaaat aacaaaaaaa catttaaaca 13020
ttagaagcct gtcttacaac aggaaaaaca acccttataa gcataagacg gactacggcc 13080
atgccggcgt gaccgtaaaa aaactggtca ccgtgattaa aaagcaccac cgacagctcc 13140
tcggtcatgt ccggagtcat aatgtaagac tcggtaaaca catcaggttg attcacatcg 13200
gtcagtgcta aaaagcgacc gaaatagccc gggggaatac atacccgcag gcgtagagac 13260
aacattacag cccccatagg aggtataaca aaattaatag gagagaaaaa cacataaaca 13320
cctgaaaaac cctcctgcct aggcaaaata gcaccctccc gctccagaac aacatacagc 13380
gcttccacag cggcagccat aacagtcagc cttaccagta aaaaagaaaa cctattaaaa 13440
aaacaccact cgacacggca ccagctcaat cagtcacagt gtaaaaaagg gccaagtgca 13500
gagcgagtat atataggact aaaaaatgac gtaacggtta aagtccacaa aaaacaccca 13560
gaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa aaaacccaca acttcctcaa 13620
atcgtcactt ccgttttccc acgttacgta acttcccatt ttaagaaaac tacaattccc 13680
aacacataca agttactccg ccctaaaacc tacgtcaccc gccccgttcc cacgccccgc 13740
gccacgtcac aaactccacc ccctcattat catattggct tcaatccaaa ataa 13794
Claims (5)
1. A recombinant plasmid based on adenovirus type 5 sequences, which is used for in vitro preparation of adeno-associated virus and comprises a sequence derived from Adv5, wherein the sequence derived from Adv5 is shown as SEQ ID No. 2.
2. The recombinant plasmid based on an adenovirus type 5 sequence of claim 1, wherein the plasmid further comprises a kanamycin resistance gene.
3. The recombinant plasmid based on adenovirus type 5 sequences according to claim 2, wherein the sequence of the plasmid is shown in SEQ ID No. 1.
4. An adeno-associated virus packaging system, comprising: a recombinant plasmid based on an adenovirus type 5 sequence according to any one of claims 1 to 3.
5. The adeno-associated virus packaging system according to claim 4 wherein the system is transfected into HEK293 cells for adeno-associated virus production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110168425.3A CN112877358B (en) | 2021-02-07 | 2021-02-07 | Recombinant plasmid based on type 5 adenovirus sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110168425.3A CN112877358B (en) | 2021-02-07 | 2021-02-07 | Recombinant plasmid based on type 5 adenovirus sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112877358A true CN112877358A (en) | 2021-06-01 |
| CN112877358B CN112877358B (en) | 2023-02-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110168425.3A Active CN112877358B (en) | 2021-02-07 | 2021-02-07 | Recombinant plasmid based on type 5 adenovirus sequence and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1278302A (en) * | 1997-11-17 | 2000-12-27 | 阿文蒂斯药物股份有限公司 | Adenovirus vectors and method for reducing homologous recombination phnomena |
| CN1816620A (en) * | 2003-05-15 | 2006-08-09 | 因特罗根治疗公司 | Methods and compositions for the production of adenoviral vectors |
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2021
- 2021-02-07 CN CN202110168425.3A patent/CN112877358B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1278302A (en) * | 1997-11-17 | 2000-12-27 | 阿文蒂斯药物股份有限公司 | Adenovirus vectors and method for reducing homologous recombination phnomena |
| CN1816620A (en) * | 2003-05-15 | 2006-08-09 | 因特罗根治疗公司 | Methods and compositions for the production of adenoviral vectors |
Non-Patent Citations (1)
| Title |
|---|
| SAMPA SANTRA ET AL.: "Replication-Defective Adenovirus Serotype 5 Vectors Elicit Durable Cellular and Humoral Immune Responses in Nonhuman Primates", 《JOURNAL OF VIROLOGY》 * |
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| CN112877358B (en) | 2023-02-03 |
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