LOCUS Exported 6770 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Modified from pCas9-CR4 (Addgene: 62655) to use the high-fidelity version of Cas9, SpCas9-HF1 (N497A/R661A/Q695A/Q926A) from Kleinstiver et al 2016.. ACCESSION . VERSION . KEYWORDS pEM-Cas9HF1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6770) AUTHORS Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar R, Lynch MD TITLE Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. JOURNAL ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174. PUBMED 28915012 REFERENCE 2 (bases 1 to 6770) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6770 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(14..637) /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind 545..564 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter 688..743 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 724..742 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 779..4882 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" /label=SpCas9-HF1 /note="carries the mutations N497A, R661A, Q695A, and Q926A" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4883..4915 /codon_start=1 /product="C-terminal peptide that mediates degradation in bacteria through the ClpXP and ClpAP proteases (McGinness et al., 2006)" /label=ssrA tag /note="wild-type LAA variant" /translation="AANDENYALAA" terminator 4942..5013 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5029..5056 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(5084..5101) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5218..5763) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(5298..5317) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5877..5971 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5992..6651) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 6566..6585 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(6652..6754) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 agatatcgac gtcttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 61 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 121 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 181 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 241 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 301 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 361 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 421 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 481 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 541 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 601 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa ttgctagcat 661 tgtacctagg actgagctag ccataaagtt gacactctat cgttgataga gttattttac 721 cactccctat cagtgataga gaaaagaatt caaaagatct aaagaggact tcggatctat 781 ggataagaaa tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat 841 cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca 901 cagtatcaaa aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc 961 gactcgcctc aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta 1021 tctacaggag attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact 1081 tgaagagtct tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa 1141 tatagtagat gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa 1201 attggtagat tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat 1261 gattaagttt cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt 1321 ggacaaacta tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat 1381 taacgcaagt ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg 1441 attagaaaat ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct 1501 cattgctttg tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga 1561 tgctaaatta cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca 1621 aattggagat caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt 1681 actttcagat atcctaagag taaatactga aataactaag gctcccctat cagcttcaat 1741 gattaaacgc tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca 1801 acaacttcca gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg 1861 ttatattgat gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga 1921 aaaaatggat ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa 1981 gcaacggacc tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc 2041 tattttgaga agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga 2101 aaaaatcttg acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg 2161 ttttgcatgg atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt 2221 tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc atgacagcgt ttgataaaaa 2281 tcttccaaat gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta 2341 taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc 2401 aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt 2461 taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc 2521 aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat 2581 taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt 2641 aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca 2701 cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggagc 2761 gttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga 2821 ttttttgaaa tcagatggtt ttgccaatcg caattttatg gcgctgatcc atgatgatag 2881 tttgacattt aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca 2941 tgaacatatt gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt 3001 aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat 3061 tgaaatggca cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat 3121 gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt 3181 tgaaaatact caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga 3241 catgtatgtg gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat 3301 tgttccacaa agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga 3361 taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa 3421 ctattggaga caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac 3481 gaaagctgaa cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt 3541 ggttgaaact cgcgcgatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac 3601 taaatacgat gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa 3661 attagtttct gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta 3721 ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata 3781 tccaaaactt gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat 3841 gattgctaag tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa 3901 tatcatgaac ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc 3961 tctaatcgaa actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc 4021 cacagtgcgc aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca 4081 gacaggcgga ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc 4141 tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta 4201 ttcagtccta gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa 4261 agagttacta gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt 4321 tttagaagct aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata 4381 tagtcttttt gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca 4441 aaaaggaaat gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca 4501 ttatgaaaag ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca 4561 gcataagcat tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat 4621 tttagcagat gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc 4681 aatacgtgaa caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc 4741 cgctgctttt aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga 4801 agttttagat gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga 4861 tttgagtcag ctaggaggtg acgctgctaa cgacgaaaac tacgctctgg ctgcttaact 4921 cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 4981 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg 5041 ggtgggcctt tctgcgttta tacctaggga tatattccgc ttcctcgctc actgactcgc 5101 tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc 5161 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt 5221 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg 5281 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc 5341 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca 5401 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg 5461 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5521 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta 5581 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct 5641 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg 5701 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct 5761 caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct 5821 tcaaatgtag cacctgaagt cagccccata cgatataagt tgttactagt gcttggattc 5881 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct 5941 gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc 6001 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc 6061 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt 6121 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa 6181 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa 6241 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg 6301 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg 6361 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt 6421 cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc 6481 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg 6541 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg 6601 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc 6661 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt 6721 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc //