LOCUS Exported 9533 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses CAS9 and sgRNA for targeted counterselection in S. aureus. ACCESSION . VERSION . KEYWORDS pCAS9counter SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9533) AUTHORS Penewit K, Holmes EA, McLean K, Ren M, Waalkes A, Salipante SJ TITLE Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection. JOURNAL MBio. 2018 Feb 20;9(1). pii: mBio.00067-18. doi: 10.1128/mBio.00067-18. PUBMED 29463653 REFERENCE 2 (bases 1 to 9533) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9533 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 1..18 /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O1 for the tetR and tetA genes" protein_bind 36..54 /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O1 for the tetR and tetA genes" CDS 77..4180 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" protein_bind 4650..4667 /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O1 for the tetR and tetA genes" misc_RNA 4798..4873 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind 7492..7514 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7552..7570) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7638..7742 /gene="bla" /label=AmpR promoter CDS 7743..8603 /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(7961..7980) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8774..9362 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9263..9282 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 actctatcat tgatagagat taattaaaat aagctactct atcattgata gagtgatcca 61 aatttaggag acaaaaatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag 121 cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct 181 gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag 241 tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg 301 gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga 361 tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg 421 tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat 481 ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta 541 tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa 601 tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt 661 atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg 721 attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa 781 tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa 841 ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt 901 agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa 961 tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc 1021 tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt 1081 aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc 1141 aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt 1201 tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg 1261 tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca 1321 cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga 1381 caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt 1441 ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc 1501 atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat 1561 gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta 1621 tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg 1681 aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac 1741 aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt 1801 tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca 1861 tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat 1921 cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag 1981 acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg 2041 ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc 2101 tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca 2161 gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg 2221 acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa 2281 aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa 2341 gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa 2401 aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat 2461 tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta 2521 tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga 2581 ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa 2641 ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt 2701 agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg 2761 taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg 2821 ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt 2881 ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt 2941 gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt 3001 acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac 3061 tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt 3121 ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa 3181 atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg 3241 agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga 3301 taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt 3361 caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa 3421 ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga 3481 tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa 3541 gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga 3601 aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat 3661 cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc 3721 tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt 3781 tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa 3841 acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga 3901 attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa 3961 caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt 4021 gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg 4081 atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct 4141 ttatgaaaca cgcattgatt tgagtcagct aggaggtgac gttgcagctt gaagtatatt 4201 ttagatgaag attatttctt aataactaaa aatatggtat aatactctta ataaatgcag 4261 taatacaggg gcttttcaag actcccgggt accgagctcg aattcaggcg cgcctattct 4321 aaatgcataa taaatactga taacatctta tattttgtat tatattttgt attatcgttg 4381 acatgtataa ttttgatatc aaaaactgat tttccctcta ttattttcga gatttatttt 4441 cttaattctc tttaacaaac tagaaatatt gtatatacaa aaaattataa ataatagatg 4501 aatagtttaa ttataggtgt tcatcaatcg aaaaagcaac gtatcttatt taaagtgcgt 4561 tgcttttttc tcatttataa ggttaaataa ttctcatata tcaagcaaag tgacaggcgc 4621 cttcatgaaa aactaaaaaa aatatgaaca ctctatcatt gatagagatt aattaaaata 4681 agctcgagac cacctgtata aaacaaatca atatagctaa ggacaaaagc gactttagat 4741 tctaactatt gattttggcg ataaagtatc atttaggaag cagatattga tggtctcgtt 4801 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 4861 accgagtcgg tgctttttta gtctgccaga gaatataaaa agccagatta ttaatccggc 4921 ttttttatta tttggcgcca tcacacgcaa aaaggaaatt ggaataaatg cgaaatttga 4981 gatgttaatt aaagaccttt ttgaggtctt tttttcttag atttttgggg ttatttaggg 5041 gagaaaacat aggggggtac tacgacctcc cccctaggtg tccattgtcc attgtccaaa 5101 caaataaata aatattgggt ttttaatgtt aaaaggttgt tttttatgtt aaagtgaaaa 5161 aaacagatgt tgggaggtac agtgatggtt gtagatagaa aagaagagaa aaaagttgct 5221 gttactttaa gacttacaac agaagaaaat gagatattaa atagaatcaa agaaaaatat 5281 aatattagca aatcagatgc aaccggtatt ctaataaaaa aatatgcaaa ggaggaatac 5341 ggtgcatttt aaacaaaaaa agatagacag cactggcatg ctgcctatct atgactaaat 5401 tttgttaaat gtattagcac cgttattata tcatgagcga aaatgtaata aaagaaactg 5461 aaaacaagaa aaattcaaga ggacgtaatt ggacatttgt tttatatcca gaatcagcaa 5521 aagccgagtg gttagagtat ttaaaagagt tacacattca atttgtagtg tctccattac 5581 atgataggga tactgataca gaagatagga tgaaaaaaga gcattatcat attctagtga 5641 tgtatgaggg taataaatct tatgaacaga taaaaataat tacagaagaa ttgaatgcga 5701 ctattccgca gattgcagga agtgtgaaag gtcttgtgag atatatgctt cacatggacg 5761 atcctaataa atttaaatat caaaaagaag atatgatagt ttatggcggt gtagatgttg 5821 atgaattatt aaagaaaaca acaacagata gatataaatt aattaaagaa atgattgagt 5881 ttattgatga acaaggaatc gtagaattta agagtttaat ggattatgca atgaagttta 5941 aatttgatga ttggttcccg cttttatgtg ataactcggc gtatgttatt caagaatata 6001 taaaatcaaa tcggtataaa tctgaccgat agattttgaa tttaagagtg tcacaagaca 6061 ctcttttttc gcaccagcga aaactggttt aagccgactg ggcccaccta ggaattgaat 6121 aagacatgct acacctccgg ataataaata tatataaacg tatatagatt tcataaagtc 6181 taacacacta gacttattta cttcgtaatt aagtcgttaa accgtgtgct ctacgaccaa 6241 aactataaaa cctttaagaa ctttcttttt ttacaagaaa aaagaaatta gataaatctc 6301 tcatatcttt tattcaataa tcgcatccga ttgcagtata aatttaacga tcactcatca 6361 tgttcatatt tatcagagct cgtgctataa ttatactaat tttataagga ggaaaaaata 6421 tgggcatttt tagtattttt gtaatcagca cagttcatta tcaaccaaac aaaaaataag 6481 tggttataat gaatcgttaa taagcaaaat tcatataacc aaattaaaga gggttataat 6541 gaacgagaaa aatataaaac acagtcaaaa ctttattact tcaaaacata atatagataa 6601 aataatgaca aatataagat taaatgaaca tgataatatc tttgaaatcg gctcaggaaa 6661 aggccatttt acccttgaat tagtaaagag gtgtaatttc gtaactgcca ttgaaataga 6721 ccataaatta tgcaaaacta cagaaaataa acttgttgat cacgataatt tccaagtttt 6781 aaacaaggat atattgcagt ttaaatttcc taaaaaccaa tcctataaaa tatatggtaa 6841 tataccttat aacataagta cggatataat acgcaaaatt gtttttgata gtatagctaa 6901 tgagatttat ttaatcgtgg aatacgggtt tgctaaaaga ttattaaata caaaacgctc 6961 attggcatta cttttaatgg cagaagttga tatttctata ttaagtatgg ttccaagaga 7021 atattttcat cctaaaccta aagtgaatag ctcacttatc agattaagta gaaaaaaatc 7081 aagaatatca cacaaagata aacaaaagta taattatttc gttatgaaat gggttaacaa 7141 agaatacaag aaaatattta caaaaaatca atttaacaat tccttaaaac atgcaggaat 7201 tgacgattta aacaatatta gctttgaaca attcttatct cttttcaata gctataaatt 7261 atttaataag taagttaagg gatgcataaa ctgcatccct taacttgttt ttcgtgtgcc 7321 tattttttgt gaatcgatta tgtcttttgc gcagtcggct taaaccagtt ttccgcggcg 7381 ctcgagcggc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 7441 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 7501 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 7561 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 7621 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 7681 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 7741 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 7801 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 7861 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 7921 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 7981 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 8041 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 8101 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 8161 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 8221 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 8281 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 8341 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 8401 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt 8461 cacgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 8521 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 8581 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 8641 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 8701 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 8761 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 8821 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 8881 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 8941 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 9001 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 9061 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 9121 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 9181 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 9241 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 9301 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 9361 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 9421 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 9481 ctggcggccg ctgcatgcct gcaggttcat gaaaaactaa aaaaaatatg aac //