质粒名称: | pCDH-EF1-copGFP-T2A-Puro |
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出品公司: | Addgene |
目录编号: | 72263 |
存储实验室: | Kazuhiro Oka |
载体骨架基本信息 |
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载体名称: | pCDH-CMV-MCS |
载体出品公司: | Systembio |
空载体大小: | 7133 bp |
载体修饰: | 将ClaI/SalI片段替换为EF1+新的MCS |
载体类型: | 哺乳动物表达,慢病毒 |
筛选标记: | 嘌呤霉素 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | copGFP-T2A-Puro |
插入大小: | 1434 bp |
启动子: | elongation factor-1 |
克隆方法: | 限制性内切酶 |
5’克隆位点: | BamHI(如果已销毁,则未知) |
3’克隆位点: | EcoRI (未破坏) |
5’测序引物 | GCACTTGATGTAATTCTCC |
3’测序引物 | CAACACCACGGAATTGTCAG |
文献引用方法 |
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材料和方法部分: | pCDH-EF1-copGFP-T2A-Puro was a gift from Kazuhiro Oka (Addgene plasmid # 72263 ; http://n2t.net/addgene:72263 ; RRID:Addgene_72263) |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pCDH-EF1-copGFP-T2A-Puro 8549 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express copGFP and puromycin resistance gene under EF1 promoter. ACCESSION . VERSION . KEYWORDS pCDH-EF1-copGFP-T2A-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8549) TITLE lenti vector JOURNAL Unpublished REFERENCE 2 (bases 1 to 8549) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8549 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 234..414 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 458..583 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1076..1309 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1493..1537 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 1804..1921 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2013..3191 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2244..3182 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3139..3159 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3231..3893 /codon_start=1 /product="green fluorescent protein 2 from Pontellina plumata, also known as ppluGFP2 (Shagin et al., 2004)" /label=CopGFP /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA" CDS 3978..4031 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4032..4631 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(4032..4051) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4528..4548 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 4644..5232 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4697..4717) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5115..5126) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 5306..5539 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 5611..5732 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5648..5667) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5702..5721 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5751..5886 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5813..5832 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5924..5940) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5924..5940) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5937..5959) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5948..5964 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5972..6002) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6017..6038 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6155..6172) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6326..6914) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6406..6425) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7085..7945) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7708..7727 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7946..8050) /gene="bla" /label=AmpR promoter primer_bind 8118..8136 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(8174..8196) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 8296..8315 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8509..8531 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8523..8540 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8524..8540 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 1921 tatcgatagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat 1981 ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc 2041 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 2101 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 2161 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 2221 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 2281 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc 2341 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag 2401 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa 2461 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt 2521 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag 2581 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc 2641 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt 2701 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc 2761 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc 2821 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg 2881 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca 2941 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt 3001 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga 3061 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt 3121 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc 3181 aggtgtcgtg agcaattgga tccaccatgg agagcgacga gagcggcctg cccgccatgg 3241 agatcgagtg ccgcatcacc ggcaccctga acggcgtgga gttcgagctg gtgggcggcg 3301 gagagggcac ccccaagcag ggccgcatga ccaacaagat gaagagcacc aaaggcgccc 3361 tgaccttcag cccctacctg ctgagccacg tgatgggcta cggcttctac cacttcggca 3421 cctaccccag cggctacgag aaccccttcc tgcacgccat caacaacggc ggctacacca 3481 acacccgcat cgagaagtac gaggacggcg gcgtgctgca cgtgagcttc agctaccgct 3541 acgaggccgg ccgcgtgatc ggcgacttca aggtggtggg caccggcttc cccgaggaca 3601 gcgtgatctt caccgacaag atcatccgca gcaacgccac cgtggagcac ctgcacccca 3661 tgggcgataa cgtgctggtg ggcagcttcg cccgcacctt cagcctgcgc gacggcggct 3721 actacagctt cgtggtggac agccacatgc acttcaagag cgccatccac cccagcatcc 3781 tgcagaacgg gggccccatg ttcgccttcc gccgcgtgga ggagctgcac agcaacaccg 3841 agctgggcat cgtggagtac cagcacgcct tcaagacccc cattgccttc gccagatccc 3901 gcgctcagtc gtccaattct gccgtggacg gcaccgccgg acccggctcc accggatctc 3961 gcaagcttgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 4021 atcctggccc aatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 4081 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 4141 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 4201 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 4261 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 4321 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 4381 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 4441 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 4501 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 4561 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 4621 ccggtgcctg agaattcgtc gacaatcaac ctctggatta caaaatttgt gaaagattga 4681 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 4741 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 4801 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 4861 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 4921 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 4981 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 5041 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 5101 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 5161 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 5221 ccgcctcccc gcctggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 5281 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aaaataagat 5341 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 5401 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 5461 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 5521 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt 5581 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac 5641 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 5701 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc 5761 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 5821 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 5881 gaggcctaga cttttgcaga gacggcccaa attcgtaatc atggtcatag ctgtttcctg 5941 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 6001 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 6061 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 6121 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 6181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 6241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 6301 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 6361 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 6421 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 6481 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 6541 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 6601 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6661 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6721 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 6781 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6841 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6901 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6961 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 7021 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 7081 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 7141 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 7201 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 7261 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 7321 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 7381 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 7441 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 7501 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 7561 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 7621 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7681 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 7741 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7801 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7861 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7921 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7981 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 8041 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 8101 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg 8161 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 8221 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 8281 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 8341 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc 8401 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 8461 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 8521 gttgtaaaac gacggccagt gccaagctg //
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