LOCUS Exported 8008 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR-cas9 targeting in E. coli with ampicillin resistance. ACCESSION . VERSION . KEYWORDS pJZ002 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8008) AUTHORS Bauman KD, Li J, Murata K, Mantovani SM, Dahesh S, Nizet V, Luhavaya H, Moore BS TITLE Refactoring the Cryptic Streptophenazine Biosynthetic Gene Cluster Unites Phenazine, Polyketide, and Nonribosomal Peptide Biochemistry. JOURNAL Cell Chem Biol. 2019 Feb 15. pii: S2451-9456(19)30038-8. doi: 10.1016/j.chembiol.2019.02.004. PUBMED 30853419 REFERENCE 2 (bases 1 to 8008) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8008 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(117..1067) /codon_start=1 /gene="rep101" /product="RepA protein needed for replication with the pSC101 origin" /label=Rep101 /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 1115..1337 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" CDS complement(1959..2819) /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 2582..2601 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2820..2924) /gene="bla" /label=AmpR promoter misc_RNA complement(3084..3162) /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 3458..7564 /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" misc_feature 7585..7716 /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 7717..7752 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 7783..7818 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" ORIGIN 1 taagctagcc catgggtatg gacagttttc cctttgatat gtaacggtga acagttgttc 61 tacttttgtt tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag 121 atccttccgt atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat 181 gagaacgaac cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa 241 ctggtgagct gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttacgt 301 aggtaggaat ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg 361 ttctcaagtt cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta 421 tcagtcgggc ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct 481 ttacttattg gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca 541 agcattaaca tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc 601 ttttgtgtta gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa 661 gacttaacat gttccagatt atattttatg aattttttta actggaaaag ataaggcaat 721 atctcttcac taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac 781 tggaaaatct caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc 841 tctctggttg ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga 901 gcgtattggt tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg 961 gggttgagta gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag 1021 cgactaatcg ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg 1081 tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc 1141 cttttccttt gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta 1201 aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt 1261 caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc 1321 agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa 1381 acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc 1441 gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat attccttttg tctccgacca 1501 tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg 1561 cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac 1621 ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat 1681 gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac 1741 cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg 1801 gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 1861 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 1921 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 1981 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 2041 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 2101 gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 2161 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 2221 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 2281 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 2341 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 2401 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 2461 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 2521 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 2581 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 2641 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 2701 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 2761 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 2821 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 2881 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 2941 gtgccacctg aagatggcag cgcctaaatg tagaatgata aaaggattaa gagattaatt 3001 tccctaaaaa tgataaaaca agcgttttga aagcgcttgt ttttttggtt tgcagtcaga 3061 gtagaataga agtatcaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg 3121 actagcctta ttttaacttg ctatgctgtt ttgaatggtt ccaacaagat tattttataa 3181 cttttataac aaataatcaa ggagaaattc aaagaaattt atcagccata aaacaatact 3241 taatactata gaatgataac aaaataaact actttttaaa agaattttgt gttataatct 3301 atttattatt aagtattggg taatattttt tgaagagata ttttgaaaaa gaaaaattaa 3361 agcatattaa actaatttcg gaggtcatta aaactattat tgaaatcatc aaactcatta 3421 tggatttaat ttaaactttt tattttagga ggcaaaaatg gataagaaat actcaatagg 3481 cttagatatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc 3541 gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat 3601 aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc 3661 tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa 3721 tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga 3781 agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta 3841 tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa 3901 agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt 3961 tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt 4021 ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg gagtagatgc 4081 taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc tcattgctca 4141 gctccccggt gagaagaaaa atggcttatt tgggaatctc attgctttgt cattgggttt 4201 gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac agctttcaaa 4261 agatacttac gatgatgatt tagataattt attggcgcaa attggagatc aatatgctga 4321 tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata tcctaagagt 4381 aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct acgatgaaca 4441 tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag aaaagtataa 4501 agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg ggggagctag 4561 ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg gtactgagga 4621 attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct ttgacaacgg 4681 ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa gacaagaaga 4741 cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga cttttcgaat 4801 tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa 4861 gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag gtgcttcagc 4921 tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg aaaaagtact 4981 accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa 5041 atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat 5101 tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta 5161 tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa 5221 tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga 5281 taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga 5341 tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat 5401 gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa 5461 tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt 5521 tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat 5581 tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc 5641 tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt 5701 caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca 5761 gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat 5821 caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa 5881 tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt 5941 agatattaat cgtttaagtg attatgatgt cgatcacatt gttccacaaa gtttccttaa 6001 agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga 6061 taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa 6121 cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt 6181 gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac 6241 taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa 6301 acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa 6361 agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta 6421 tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt 6481 tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga 6541 aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac 6601 agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga 6661 aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc 6721 catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga 6781 gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc 6841 aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa 6901 ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat 6961 tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa 7021 ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa 7081 cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct 7141 gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag 7201 tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga 7261 gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga 7321 taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa 7381 tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga 7441 tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat 7501 ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga 7561 ctgaagtata ttttagatga agattatttc ttaataacta aaaatatggt ataatactct 7621 taataaatgc agtaatacag gggcttttca agactgaagt ctagctgaga caaatagtgc 7681 gattacgaaa ttttttagac aaaaatagtc tacgaggttt tagagctatg ctgttttgaa 7741 tggtcccaaa actgagacca gtctcggaag ctcaaaggtc tcgttttaga gctatgctgt 7801 tttgaatggt cccaaaactt cagcacactg agacttgttg agttccatgt tttagagcta 7861 tgctgttttg aatggactcc attcaacatt gccgatgata acttgagaaa gagggttaat 7921 accagcagtc ggataccttc ctattctttc tgttaaagcg ttttcatgtt ataataggca 7981 aaagaagagt agtgtgatcg tccattcc //