LOCUS       sequence-213782-        8183 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 8183)
  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 8183)
  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..8183
                     /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(4905..4922)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5039..5584)
                     /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(5119..5138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5698..5792)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5813..6472)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6387..6406
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6473..6575)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6605..7231)
                     /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        7247..7302
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7283..7301
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             7320..7331
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(7338..7364,7365..7367,7368..8183,1..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 ggggtttttt cgccctaggg atatattccg cttcctcgct cactgactcg
     4921 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     4981 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt
     5041 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     5101 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     5161 ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     5221 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     5281 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5341 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     5401 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     5461 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     5521 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     5581 tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc aatttatctc
     5641 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgttactag tgcttggatt
     5701 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
     5761 tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgatatca aattacgccc
     5821 cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc
     5881 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     5941 tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta
     6001 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa
     6061 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt
     6121 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt
     6181 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt
     6241 tcattgccat acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg
     6301 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct
     6361 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac
     6421 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt
     6481 ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat
     6541 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc cagatatcga
     6601 cgtcttaaga cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa
     6661 ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta
     6721 ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct
     6781 cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg
     6841 cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat
     6901 actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta
     6961 aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta
     7021 aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag
     7081 cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga
     7141 gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa
     7201 tcactttact tttatctaat ctagacatca ttaattccta atttttgttg acactctatc
     7261 gttgatagag ttattttacc actccctatc agtgatagag aaaagaattc aaaagatcta
     7321 aagaggagaa aggatctatg gataagaaat actcaatagg cttagctatc ggcacaaata
     7381 gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc
     7441 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca
     7501 gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc
     7561 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg
     7621 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac
     7681 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta
     7741 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct
     7801 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa
     7861 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat
     7921 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac
     7981 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa
     8041 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa
     8101 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt
     8161 tagataattt attggcgcaa att
//
