LOCUS Exported 5292 bp ds-DNA linear SYN 13-JUN-2021 DEFINITION synthetic linear DNA ACCESSION . VERSION . KEYWORDS pMJ826 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5292) AUTHORS Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E TITLE A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. JOURNAL Science. 2012 Jun 28. PUBMED 22745249 REFERENCE 2 (bases 1 to 5292) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5292 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 4..24 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" CDS 28..1125 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" primer_bind 1099..1122 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1156..1176 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1186..5292 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(H840A) /note="generates RNA-guided single strand nicks in DNA due to the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" ORIGIN 1 atgcatcacc atcaccatca ccatggaaaa atcgaagaag gtaaactggt aatctggatt 61 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc 121 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg 181 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct 241 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg 301 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa 361 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag 421 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg 481 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat 541 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt 601 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc 661 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca 721 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag 781 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg 841 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa 901 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg 961 gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg 1021 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc 1081 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac 1141 aacaacaaca ctagtgaaaa cctgtatttc cagggagcag cctcgatgga taagaaatac 1201 tcaataggct tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat 1261 aaggttccgt ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa 1321 aatcttatag gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa 1381 cggacagctc gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt 1441 ttttcaaatg agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt 1501 ttggtggaag aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa 1561 gttgcttatc atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct 1621 actgataaag cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt 1681 ggtcattttt tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt 1741 atccagttgg tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga 1801 gtagatgcta aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc 1861 attgctcagc tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca 1921 ttgggtttga cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag 1981 ctttcaaaag atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa 2041 tatgctgatt tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc 2101 ctaagagtaa atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac 2161 gatgaacatc atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa 2221 aagtataaag aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg 2281 ggagctagcc aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt 2341 actgaggaat tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt 2401 gacaacggct ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga 2461 caagaagact tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact 2521 tttcgaattc cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg 2581 actcggaagt ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt 2641 gcttcagctc aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa 2701 aaagtactac caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca 2761 aaggtcaaat atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag 2821 aaagccattg ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa 2881 gaagattatt tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat 2941 agatttaatg cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat 3001 tttttggata atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta 3061 tttgaagata gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat 3121 aaggtgatga aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa 3181 ttgattaatg gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca 3241 gatggttttg ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa 3301 gaagacattc aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca 3361 aatttagctg gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat 3421 gaattggtca aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt 3481 gaaaatcaga caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa 3541 gaaggtatca aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa 3601 ttgcaaaatg aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac 3661 caagaattag atattaatcg tttaagtgat tatgatgtcg atgccattgt tccacaaagt 3721 ttccttaaag acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt 3781 aaatcggata acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa 3841 cttctaaacg ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt 3901 ggaggtttga gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc 3961 caaatcacta agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa 4021 aatgataaac ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac 4081 ttccgaaaag atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat 4141 gatgcgtatc taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa 4201 tcggagtttg tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct 4261 gagcaagaaa taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc 4321 ttcaaaacag aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact 4381 aatggggaaa ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa 4441 gtattgtcca tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc 4501 tccaaggagt caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac 4561 tgggatccaa aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg 4621 gttgctaagg tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg 4681 atcacaatta tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa 4741 ggatataagg aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag 4801 ttagaaaacg gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag 4861 ctggctctgc caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg 4921 aagggtagtc cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat 4981 ttagatgaga ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc 5041 aatttagata aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa 5101 gcagaaaata ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa 5161 tattttgata caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc 5221 actcttatcc atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta 5281 ggaggtgact ga //