质粒名称: | lenti sgRNA(MS2)_puro optimized backbone |
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出品公司: | 爱迪基因 |
目录编号: | 73797 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | pLKO |
载体抗性: | 氨苄青霉素 |
空载体大小: | 8162 bp |
载体类型: | 哺乳动物表达,慢病毒,CRISPR |
筛选标记: | 嘌呤霉素 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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启动子 | U6 and EF1A |
克隆方法: | 限制性内切酶 |
5' 测序引物 | GAG GGC CTA TTT CCC ATG ATT CCT TCA TAT |
3' 测序引物 | cctagaaggtccattagctgcaaagattcc |
文献引用方法 |
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材料和方法部分: | lenti sgRNA(MS2)_puro optimized backbone was a gift from Feng Zhang (Addgene plasmid # 73797 ; http://n2t.net/addgene:73797 ; RRID:Addgene_73797) |
参考文献部分: | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F. Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. Epub 2017 Mar 23. 10.1038/nprot.2017.016 PubMed 28333914 |
买家导航
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 lenti sgRNA(MS2)_puro optimized backbone 8388 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION optimized lenti sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2 and EF1a-puro resistance marker. Contains BsmBI sites for insertion of spacer sequences.. ACCESSION . VERSION . KEYWORDS lenti sgRNA(MS2)_puro optimized backbone SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8388) AUTHORS Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F TITLE Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. JOURNAL Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. Epub 2017 Mar 23. PUBMED 28333914 REFERENCE 2 (bases 1 to 8388) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8388 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 119..352 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 430..551 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(467..486) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 521..540 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 591..726 /label=SV40 ori /note="SV40 origin of replication" primer_bind 653..672 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(746..765) /label=T7 /note="T7 promoter, forward primer" promoter complement(747..765) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(775..792) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(775..791) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(784..806) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 933..1388 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1020..1039) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1230..1251 /label=F1ori-F /note="F1 origin, forward primer" promoter 1414..1518 /gene="bla" /label=AmpR promoter CDS 1519..2379 /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 complement(1737..1756) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2550..3138 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3039..3058 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3292..3309 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3426..3447 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3462..3492 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3500..3516 /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)." primer_bind 3505..3527 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3524..3540 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3524..3540 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3559..3579 /label=T3 /note="T3 promoter, forward primer" promoter 3561..3579 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3607..3833 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 3834..4014 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 4061..4186 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 4679..4912 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." promoter 5319..5559 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 5319..5339 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 5490..5509 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 5605..5623 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 5675..5693 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_feature 5785..5902 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 6041..7219 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 6272..7210 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 7167..7187 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 7232..7831 /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(7232..7251) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7728..7748 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 7847..47 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(7900..7920) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8318..8329) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" ORIGIN 1 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga 61 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 121 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 181 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 241 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 301 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat 361 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag 421 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 481 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 541 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc 601 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 661 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 721 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc 781 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 841 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 901 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 961 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 1021 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 1081 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 1141 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 1201 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 1261 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 1321 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 1381 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 1441 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 1501 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 1561 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 1621 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 1681 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 1741 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 1801 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 1861 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1921 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1981 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 2041 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 2101 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 2161 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 2221 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 2281 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 2341 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 2401 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 2461 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 2521 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2581 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2641 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 2701 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2761 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2821 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2881 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2941 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 3001 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 3061 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 3121 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 3181 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 3241 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 3301 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 3361 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 3421 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 3481 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 3541 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa 3601 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc 3661 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 3721 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 3781 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc 3841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 3901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 3961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 4021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 4081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 4141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 4201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 4261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 4321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 4381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 4441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 4501 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 4561 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 4621 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 4681 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 4741 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 4801 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc 4861 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 4921 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 4981 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 5041 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga 5101 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 5161 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 5221 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 5281 cgtttcagac ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc 5341 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg 5401 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 5461 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 5521 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacacccga gacgggatac 5581 cgtctctgtt ttagagctag gccaacatga ggatcaccca tgtctgcagg gcctagcaag 5641 ttaaaataag gctagtccgt tatcaacttg gccaacatga ggatcaccca tgtctgcagg 5701 gccaagtggc accgagtcgg tgcttttttg gatccaagct tggcgtaact agatcttgag 5761 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc 5821 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca 5881 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg 5941 ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga ggaatctttg 6001 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg 6061 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc 6121 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 6181 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 6241 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc 6301 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta 6361 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc 6421 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg 6481 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat 6541 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc 6601 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg 6661 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg 6721 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat 6781 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg 6841 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg 6901 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg 6961 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag 7021 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg 7081 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct 7141 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct 7201 tccatttcag gtgtcgtgac gtacggccac catgaccgag tacaagccca cggtgcgcct 7261 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 7321 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 7381 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 7441 cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 7501 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 7561 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 7621 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 7681 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 7741 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 7801 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 7861 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 7921 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 7981 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 8041 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 8101 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 8161 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 8221 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 8281 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 8341 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcg //