LOCUS Exported 6770 bp ds-DNA circular SYN 03-DEC-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6770) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 3, 2018 from SnapGene Viewer 4.2.6 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6770 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(7..630) /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" promoter 681..736 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 717..735 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 772..4911 /codon_start=1 /label=Cas9-NG /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVL DATLIHQSITGLYETRIDLSQLGGDAANDENYALAA" CDS 4876..4908 /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 4935..5006 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5022..5049 /label=T7Te terminator /note="phage T7 early transcription terminator" rep_origin complement(5211..5756) /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." terminator 5870..5964 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5985..6644) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" promoter complement(6645..6747) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 601 aatcacttta cttttatcta atctagacat cattaattcc taattgctag cattgtacct 661 aggactgagc tagccataaa gttgacactc tatcgttgat agagttattt taccactccc 721 tatcagtgat agagaaaaga attcaaaaga tctaaagagg acttcggatc tatggataag 781 aaatactcaa taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat 841 gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc 901 aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgga agcgactcgt 961 ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttg ttatctacag 1021 gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag 1081 tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta 1141 gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta 1201 gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag 1261 tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa 1321 ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca 1381 agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa 1441 aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct 1501 ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa 1561 ttacagcttt caaaagatac ttacgatgat gatttagata atttattggc gcaaattgga 1621 gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca 1681 gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa 1741 cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt 1801 ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt 1861 gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg 1921 gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg 1981 acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg 2041 agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc 2101 ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca 2161 tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat 2221 aaaggtgctt cagctcaatc atttattgaa cgcatgacaa actttgataa aaatcttcca 2281 aatgaaaaag tactaccaaa acatagtttg ctttatgagt attttacggt ttataacgaa 2341 ttgacaaagg tcaaatatgt tactgaagga atgcgaaaac cagcatttct ttcaggtgaa 2401 cagaagaaag ccattgttga tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa 2461 ttaaaagaag attatttcaa aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt 2521 gaagatagat ttaatgcttc attaggtacc taccatgatt tgctaaaaat tattaaagat 2581 aaagattttt tggataatga agaaaatgaa gatatcttag aggatattgt tttaacattg 2641 accttatttg aagataggga gatgattgag gaaagactta aaacatatgc tcacctcttt 2701 gatgataagg tgatgaaaca gcttaaacgt cgccgttata ctggttgggg acgtttgtct 2761 cgaaaattga ttaatggtat tagggataag caatctggca aaacaatatt agattttttg 2821 aaatcagatg gttttgccaa tcgcaatttt atgcagctga tccatgatga tagtttgaca 2881 tttaaagaag acattcaaaa agcacaagtg tctggacaag gcgatagttt acatgaacat 2941 attgcaaatt tagctggtag ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt 3001 gttgatgaat tggtcaaagt aatggggcgg cataagccag aaaatatcgt tattgaaatg 3061 gcacgtgaaa atcagacaac tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga 3121 atcgaagaag gtatcaaaga attaggaagt cagattctta aagagcatcc tgttgaaaat 3181 actcaattgc aaaatgaaaa gctctatctc tattatctcc aaaatggaag agacatgtat 3241 gtggaccaag aattagatat taatcgttta agtgattatg atgtcgatca cattgttcca 3301 caaagtttcc ttaaagacga ttcaatagac aataaggtct taacgcgttc tgataaaaat 3361 cgtggtaaat cggataacgt tccaagtgaa gaagtagtca aaaagatgaa aaactattgg 3421 agacaacttc taaacgccaa gttaatcact caacgtaagt ttgataattt aacgaaagct 3481 gaacgtggag gtttgagtga acttgataaa gctggtttta tcaaacgcca attggttgaa 3541 actcgccaaa tcactaagca tgtggcacaa attttggata gtcgcatgaa tactaaatac 3601 gatgaaaatg ataaacttat tcgagaggtt aaagtgatta ccttaaaatc taaattagtt 3661 tctgacttcc gaaaagattt ccaattctat aaagtacgtg agattaacaa ttaccatcat 3721 gcccatgatg cgtatctaaa tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa 3781 cttgaatcgg agtttgtcta tggtgattat aaagtttatg atgttcgtaa aatgattgct 3841 aagtctgagc aagaaatagg caaagcaacc gcaaaatatt tcttttactc taatatcatg 3901 aacttcttca aaacagaaat tacacttgca aatggagaga ttcgcaaacg ccctctaatc 3961 gaaactaatg gggaaactgg agaaattgtc tgggataaag ggcgagattt tgccacagtg 4021 cgcaaagtat tgtccatgcc ccaagtcaat attgtcaaga aaacagaagt acagacaggc 4081 ggattctcca aggagtcaat tcgaccaaaa agaaattcgg acaagcttat tgctcgtaaa 4141 aaagactggg atccaaaaaa atatggtggt tttgttagtc caacggtagc ttattcagtc 4201 ctagtggttg ctaaggtgga aaaagggaaa tcgaagaagt taaaatccgt taaagagtta 4261 ctagggatca caattatgga aagaagttcc tttgaaaaaa atccgattga ctttttagaa 4321 gctaaaggat ataaggaagt taaaaaagac ttaatcatta aactacctaa atatagtctt 4381 tttgagttag aaaacggtcg taaacggatg ctggctagtg cccgattctt acaaaaagga 4441 aatgagctgg ctctgccaag caaatatgtg aattttttat atttagctag tcattatgaa 4501 aagttgaagg gtagtccaga agataacgaa caaaaacaat tgtttgtgga gcagcataag 4561 cattatttag atgagattat tgagcaaatc agtgaatttt ctaagcgtgt tattttagca 4621 gatgccaatt tagataaagt tcttagtgca tataacaaac atagagacaa accaatacgt 4681 gaacaagcag aaaatattat tcatttattt acgttgacga atcttggagc tccccgtgct 4741 tttaaatatt ttgatacaac aattgatcgt aaagtatatc ggtctacaaa agaagtttta 4801 gatgccactc ttatccatca atccatcact ggtctttatg aaacacgcat tgatttgagt 4861 cagctaggag gtgacgctgc taacgacgaa aactacgctc tggctgctta actcgagtaa 4921 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 4981 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc 5041 ctttctgcgt ttatacctag ggatatattc cgcttcctcg ctcactgact cgctacgctc 5101 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga 5161 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg 5221 ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg 5281 acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt 5341 cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg 5401 cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc 5461 cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag 5521 acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga 5581 agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa 5641 gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca 5701 aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag 5761 atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg 5821 tagcacctga agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa 5881 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca 5941 ttactggatc tatcaacagg agtccaagcg agctcgatat caaattacgc cccgccctgc 6001 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa 6061 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat 6121 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa 6181 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta 6241 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac 6301 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg 6361 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc 6421 atacgaaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa 6481 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc 6541 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat 6601 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct 6661 cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga 6721 aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccagatatc //