质粒名称: | pU6-Decoy |
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出品公司: | 爱迪基因 |
目录编号: | 80324 |
存储实验室: | Sebastian Lourido |
载体骨架基本信息 |
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载体名称: | pU6-Universal |
载体抗性: | 氨苄青霉素 |
空载体大小: | 8360 bp |
完整质粒大小: | 8380 bp |
载体修饰: | 诱饵原间隔区已被克隆到BsaI位点。 |
载体类型: | CRISPR |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Decoy sgRNA |
启动子 | U6 |
克隆方法: | Gibson克隆 |
5' 测序引物 | GCCCATGGGATGAGACAAAG |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pU6-Decoy was a gift from Sebastian Lourido (Addgene plasmid # 80324 ; http://n2t.net/addgene:80324 ; RRID:Addgene_80324) |
参考文献部分: | A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Sidik SM, Huet D, Ganesan SM, Huynh MH, Wang T, Nasamu AS, Thiru P, Saeij JP, Carruthers VB, Niles JC, Lourido S. Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. 10.1016/j.cell.2016.08.019 PubMed 27594426 |
买家导航
pU6Decoy载体基本信息
pU6Decoy载体图谱质粒图谱和多克隆位点信息
pU6Decoy载体简介
pU6Decoy载体序列
LOCUS pU6-Decoy 8380 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Encodes Cas9 and a CRISPR sgRNA that alleviates toxicity to Cas9 in Toxoplasma gondii. ACCESSION . VERSION . KEYWORDS pU6-Decoy SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8380) AUTHORS Sidik SM, Huet D, Ganesan SM, Huynh MH, Wang T, Nasamu AS, Thiru P, Saeij JP, Carruthers VB, Niles JC, Lourido S TITLE A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. JOURNAL Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. PUBMED 27594426 REFERENCE 2 (bases 1 to 8380) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8380 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 514..579 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 586..606 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 631..4731 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 4732..4779 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(4807..4824) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4813..5020 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5029..5169 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5029..5158 /label=AAV2 ITR rep_origin 5244..5699 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5331..5350) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5541..5562 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5716..5735) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5835..5857 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5895..5913) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5981..6085 /gene="bla" /label=AmpR promoter CDS 6086..6946 /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(6304..6323) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7117..7705 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7606..7625 /label=pBR322ori-F /note="pBR322 origin, forward primer" misc_RNA 8288..8363 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 ctagaggtac catgcatgtc ccgcgttcgt gaaattctct gcatcagcgg agtgatcagg 61 aatcatcgtc tcagcgggat gacgttgcgg agcaggccgg ctcgcggtgg gcagtcagat 121 gccgaaggcg taactcagga cggcttgcgc tcatcgcaga acaggggtgg tgcctgcatt 181 gggtgcggtt ggtgatcctg gttggaccgg tggagatgcg cgcgcacgaa ggggatgtgt 241 cagaaacatt ttgtttgttc tctgtgaact tttagatgtg ttaaaggcgg cgaatattag 301 cagagagtcc tccttgttgg attctctctt gaatttcgcc ctttccttct ctttgcgagt 361 ctcgtagaga acaagcactc gttcgccgtc cctgacgacg caacccgcgc agaagacatc 421 caccaaacgg tgttacacaa tcaccttgtg tgaagttctt gcggaaaact actcgttggc 481 attttttctt gaattccctt tttcgacacc atggactata aggaccacga cggagactac 541 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg 601 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc 661 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa 721 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg 781 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga 841 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc 901 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag 961 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag 1021 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg 1081 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag 1141 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc 1201 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc 1261 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc 1321 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac 1381 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac 1441 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg 1501 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 1561 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 1621 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc 1681 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag 1741 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 1801 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc 1861 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca 1921 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac 1981 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 2041 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc 2101 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 2161 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc 2221 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 2281 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 2341 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg 2401 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 2461 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 2521 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 2581 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 2641 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 2701 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 2761 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 2821 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2881 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 2941 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg 3001 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 3061 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac 3121 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 3181 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc 3241 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg 3301 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg 3361 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg 3421 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg 3481 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3541 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc 3601 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 3661 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag 3721 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc 3781 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag 3841 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa 3901 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 3961 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac 4021 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag 4081 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 4141 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa 4201 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag 4261 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa 4321 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat 4381 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag 4441 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg 4501 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac 4561 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc 4621 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 4681 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg 4741 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct 4801 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 4861 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 4921 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 4981 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag 5041 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 5101 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct 5161 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 5221 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 5281 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 5341 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 5401 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 5461 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 5521 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 5581 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 5641 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt 5701 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 5761 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 5821 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 5881 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 5941 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 6001 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 6061 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 6121 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 6181 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 6241 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 6301 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 6361 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 6421 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 6481 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 6541 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 6601 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6661 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 6721 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6781 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa 6841 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6901 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 6961 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 7021 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 7081 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 7141 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 7201 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 7261 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 7321 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 7381 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 7441 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 7501 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 7561 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 7621 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 7681 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 7741 cttttgctgg ccttttgctc acatgggatg agacaaagtg cgcgagttga aatcgtcgtg 7801 gggacgattt caccgcggcc acatgttgga gacactgagg gcacacggga aacgcgaaag 7861 atttcaaatt aacgtaccca aacgcgaaag cttgcgcagc atacactcga agcgaacatc 7921 ccgaaccatc gagaggcgga gagcgataag tctttcacgc tgcgaagtgt tgcgacggct 7981 gcgccgctgc actgtgaatt gggcgccaat attgcatcct aggcctgacg cgcctcctgc 8041 agaacgcgag acactgggat atgtagagcc aagggggaaa ccttcgaact ctcgaatgtc 8101 ttctctgaca agaatcatat ttccatcagt tctgtcagat tttcaaatgg cgacctgcag 8161 aggcctgctt cctccctgtg cgctcttcga aggggctttc tgtcgcgcag ggtcacctcg 8221 tccccgaagg gggtgtttgc cttctggtaa atggggatgt caagttgaga atgcagttta 8281 gcaccgtgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg 8341 aaaaagtggc accgagtcgg tgcttttttt ttctttttct //