LOCUS Exported 7011 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expressing evolved CasTA of dCas9 fused AsiA variant 1.1. ACCESSION . VERSION . KEYWORDS pdCas9- AsiA_m1.1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7011) AUTHORS Ho HI, Fang JR, Cheung J, Wang HH TITLE Programmable CRISPR-Cas transcriptional activation in bacteria. JOURNAL Mol Syst Biol. 2020 Jul;16(7):e9427. doi: 10.15252/msb.20199427. PUBMED 32657546 REFERENCE 2 (bases 1 to 7011) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7011 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..56 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 37..55 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" RBS 74..85 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 92..4195 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" terminator 4528..4599 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4615..4642 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(4670..4687) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4804..5349) /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(4884..4903) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5463..5557 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5578..6237) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 6152..6171 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(6238..6340) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS complement(6370..6993) /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 6901..6920 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" ORIGIN 1 gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat agagaaaaga 61 attcaaaaga tctaaagagg agaaaggatc tatggataag aaatactcaa taggcttagc 121 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa 181 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc 241 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag 301 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat 361 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga 421 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga 481 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga 541 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat 601 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca 661 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc 721 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc 781 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc 841 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac 901 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt 961 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac 1021 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca 1081 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat 1141 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga 1201 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt 1261 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat 1321 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta 1381 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta 1441 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga 1501 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc 1561 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa 1621 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt 1681 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga 1741 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa 1801 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc 1861 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga 1921 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga 1981 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca 2041 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat 2101 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa 2161 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa 2221 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag 2281 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt 2341 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac 2401 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga 2461 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa 2521 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat 2581 taatcgttta agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga 2641 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt 2701 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa 2761 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga 2821 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca 2881 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat 2941 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt 3001 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa 3061 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta 3121 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg 3181 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat 3241 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg 3301 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc 3361 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat 3421 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa 3481 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga 3541 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga 3601 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt 3661 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg 3721 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag 3781 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga 3841 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat 3901 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt 3961 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat 4021 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac 4081 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca 4141 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactgcgc 4201 aggtggtggc ggttctggtg gtggtggcag catgaacaag aacatcgaca cggtccgtga 4261 aatcattaca gtagcttcca ttctgatcaa gttttcccgc gaggatatcg ttgaaaaccg 4321 tgcgaacttt attgccttct tgaacgaaat tggagttact cacgaggggc gcaagttgaa 4381 tcagaactcg tttcgcaaga ttataagcaa actgacgcag gaagacaaaa aaactttaat 4441 tgatgagttc aacgagggat ttgagggcgt ttatcgttat ttggaaatgt ataccaataa 4501 gtaacccgag taaggatctc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 4561 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca 4621 ccttcgggtg ggcctttctg cgtttatacc tagggatata ttccgcttcc tcgctcactg 4681 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag 4741 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc 4801 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg 4861 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt 4921 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt 4981 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta 5041 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt 5101 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag 5161 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac 5221 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa 5281 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa 5341 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt 5401 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgctt 5461 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg 5521 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta 5581 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 5641 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 5701 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac 5761 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 5821 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 5881 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 5941 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 6001 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat 6061 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 6121 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 6181 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt 6241 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat 6301 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat 6361 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa 6421 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg 6481 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg 6541 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata 6601 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt 6661 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact 6721 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc 6781 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag 6841 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg 6901 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct 6961 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt t //