LOCUS       Exported                6705 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  aTc-inducible expression of a catalytically inactive bacterial Cas9 
            (S. pyogenes) for bacterial gene knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    pdCas9-bacteria
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 6705)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6705
                     /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"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             740..4846
                     /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      4870..4941
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4957..4984
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5012..5029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5691)
                     /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(5226..5245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5805..5899
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5920..6579)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6494..6513
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6580..6682)
                     /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 taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taaagaggag aaaggatcta tggataagaa atactcaata ggcttagcta tcggcacaaa
      781 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
      841 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
      901 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
      961 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
     1021 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
     1081 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
     1141 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat
     1201 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
     1261 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
     1321 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
     1381 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
     1441 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
     1501 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
     1561 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
     1621 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
     1681 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
     1741 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
     1801 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
     1861 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
     1921 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat
     1981 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
     2041 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
     2101 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
     2161 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
     2221 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
     2281 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
     2341 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
     2401 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
     2461 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
     2521 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
     2581 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
     2641 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
     2701 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca
     2761 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
     2821 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
     2881 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
     2941 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
     3001 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
     3061 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
     3121 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
     3181 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
     3241 tgattatgat gtcgatgcca ttgttccaca aagtttcctt aaagacgatt caatagacaa
     3301 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
     3361 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
     3421 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
     3481 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat
     3541 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
     3601 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
     3661 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
     3721 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
     3781 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
     3841 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
     3901 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
     3961 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
     4021 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
     4081 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
     4141 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
     4201 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
     4261 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
     4321 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
     4381 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
     4441 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
     4501 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
     4561 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
     4621 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
     4681 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
     4741 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
     4801 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaactcg agtaaggatc
     4861 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     4921 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     4981 tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
     5041 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
     5101 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
     5161 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
     5221 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
     5281 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
     5341 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
     5401 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
     5461 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
     5521 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
     5581 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
     5641 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
     5701 cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca
     5761 cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc accaataaaa
     5821 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact
     5881 ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc cctgccactc
     5941 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     6001 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     6061 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     6121 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     6181 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     6241 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     6301 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     6361 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     6421 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     6481 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     6541 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     6601 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
     6661 ggaacctctt acgtgccgat caacgtctca ttttcgccag atatc
//
