LOCUS       sequence-217364-        8182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTet-dCas9, pTet-MCP-SoxS_R93A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8182)
  AUTHORS   Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG
  TITLE     Synthetic CRISPR-Cas gene activators for transcriptional 
            reprogramming in bacteria.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2489. doi: 10.1038/s41467-018-04901-6.
  PUBMED    29950558
REFERENCE   2  (bases 1 to 8182)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-06-27"; volume: "9(1):2489. doi"; pages: " 
            10.1038/s41467-018-04901-6"
COMMENT     Sequence Label: pJF104B
FEATURES             Location/Qualifiers
     source          1..8182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3285..3356
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3372..3399
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3417..4043)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        4059..4114
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4095..4113
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4126..4137
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(4904..4921)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5038..5583)
                     /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(5118..5137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5697..5791)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5812..6471)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6386..6405
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6472..6574)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6604..7230)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        7246..7301
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7282..7300
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             7319..7330
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(7337..7363,7364..7366,7367..8182,1..1671,1672..1674,
                     1675..3261)
                     /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"
ORIGIN
        1 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
       61 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
      121 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
      181 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
      241 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
      301 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
      361 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
      421 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
      481 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
      541 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
      601 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
      661 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
      721 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
      781 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
      841 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
      901 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
      961 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
     1021 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
     1081 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
     1141 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
     1201 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
     1261 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
     1321 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
     1381 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
     1441 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
     1501 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
     1561 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
     1621 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tgccattgtt
     1681 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
     1741 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
     1801 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
     1861 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
     1921 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
     1981 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
     2041 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
     2101 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
     2161 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
     2221 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
     2281 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
     2341 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
     2401 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
     2461 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
     2521 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
     2581 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
     2641 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
     2701 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
     2761 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
     2821 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
     2881 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
     2941 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
     3001 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
     3061 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
     3121 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
     3181 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
     3241 agtcagctag gaggtgacta actcgagtaa ggatctccag gcatcaaata aaacgaaagg
     3301 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     3361 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag ctgcagttaa
     3421 gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc aaggccgaat
     3481 aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg taataatggc
     3541 ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg ctcttgatct
     3601 tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa tgcattctct
     3661 agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc atactgtttt
     3721 tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag taaagcacat
     3781 ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc taaagggcaa
     3841 aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa agcccgctta
     3901 ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc gagtttacgg
     3961 gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt aatcacttta
     4021 cttttatcta atctagacat cattaattcc taatttttgt tgacactcta tcgttgatag
     4081 agttatttta ccactcccta tcagtgatag agaaaagaat tcattaaaga ggagaaaggt
     4141 accatggggc ccgcttctaa ctttactcag ttcgttctcg tcgacaatgg cggaactggc
     4201 gacgtgactg tcgccccaag caacttcgct aacgggatcg ctgaatggat cagctctaac
     4261 tcgcgttcac aggcttacaa agtaacctgt agcgttcgtc agagctctgc gcagaatcgc
     4321 aaatacacca tcaaagtcga ggtgcctaaa ggcgcctggc gttcgtactt aaatatggaa
     4381 ctaaccattc caattttcgc cacgaattcc gactgcgagc ttattgttaa ggcaatgcaa
     4441 ggtctcctaa aagatggaaa cccgattccc tcagcaatcg cagcaaactc cggcatctac
     4501 ggtggcggag gtagcatgtc ccatcagaaa attattcagg atcttatcgc atggattgac
     4561 gagcatattg accagccgct taacattgat gtagtcgcaa aaaaatcagg ctattcaaag
     4621 tggtacttgc aacgaatgtt ccgcacggtg acgcatcaga cgcttggcga ttacattcgc
     4681 caacgccgcc tgttactggc cgccgttgag ttgcgcacca ccgagcgtcc gatttttgat
     4741 atcgcaatgg acctgggtta tgtctcgcag cagaccttct cccgcgtttt cgcgcggcag
     4801 tttgatcgca ctcccagcga ttatcgccac cgcctgtaag cggccgccac gcaaaaaacc
     4861 ccgcttcggc gggttttttc gccctaggga tatattccgc ttcctcgctc actgactcgc
     4921 tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc
     4981 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt
     5041 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg
     5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc
     5161 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca
     5221 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg
     5281 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5341 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta
     5401 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct
     5461 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg
     5521 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct
     5581 caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct
     5641 tcaaatgtag cacctgaagt cagccccata cgatataagt tgttactagt gcttggattc
     5701 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     5761 gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc
     5821 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     5881 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     5941 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     6001 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     6061 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     6121 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     6181 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     6241 cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     6301 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     6361 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     6421 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     6481 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     6541 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agatatcgac
     6601 gtcttaagac ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag
     6661 gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa
     6721 taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc
     6781 ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc
     6841 attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata
     6901 ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa
     6961 agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa
     7021 agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc
     7081 ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag
     7141 tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat
     7201 cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga cactctatcg
     7261 ttgatagagt tattttacca ctccctatca gtgatagaga aaagaattca aaagatctaa
     7321 agaggagaaa ggatctatgg ataagaaata ctcaataggc ttagctatcg gcacaaatag
     7381 cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct
     7441 gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag
     7501 tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg
     7561 gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga
     7621 tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg
     7681 tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat
     7741 ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta
     7801 tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa
     7861 tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt
     7921 atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg
     7981 attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa
     8041 tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa
     8101 ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt
     8161 agataattta ttggcgcaaa tt
//
