LOCUS       sequence-439389-        8054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8054)
  AUTHORS   Santos-Moreno J, Tasiudi E, Stelling J, Schaerli Y
  TITLE     Multistable and dynamic CRISPRi-based synthetic circuits.
  JOURNAL   Nat Commun. 2020 Jun 2;11(1):2746. doi: 10.1038/s41467-020-16574-1.
  PUBMED    32488086
REFERENCE   2  (bases 1 to 8054)
  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: "2020-06-2"; volume: "11(1):2746. doi"; pages: " 
            10.1038/s41467-020-16574-1"
COMMENT     Sequence Label: pJ1996_v2
FEATURES             Location/Qualifiers
     source          1..8054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     162..179
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     163..179
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        213..246
                     /label=BBa_J23104
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.72"
     RBS             317..328
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(335..361,362..364,365..2851,2852..2854,2855..4441)
                     /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"
     promoter        5061..5095
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        5061..5095
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        5061..5095
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     RBS             5165..5176
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5183..5746
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     primer_bind     complement(5916..5934)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5930..5977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5986..6072
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6164..6191
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(6320..7111)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDDGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(7112..7203)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
       61 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
      121 aggagctgac tgggttgctc ctaggatgca gtgactcgag ttgtaaaacg acggccagtc
      181 aattggaatt cgagctccag cctgcggtcc ggttgacagc tagctcagtc ctaggtattg
      241 tactagttcg ctgggacgcc cgagtgacga ctgcgaagta acctctattt atcagcctgc
      301 ggtccggggt accattaaag aggagaaaga attcatggat aagaaatact caataggctt
      361 agctatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc
      421 taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg
      481 ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg
      541 tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga
      601 gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga
      661 agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca
      721 tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc
      781 ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt
      841 gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt
      901 acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa
      961 agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct
     1021 ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac
     1081 ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga
     1141 tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt
     1201 gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa
     1261 tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca
     1321 tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga
     1381 aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca
     1441 agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt
     1501 attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc
     1561 tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt
     1621 ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc
     1681 ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc
     1741 tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca
     1801 atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc
     1861 aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata
     1921 tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt
     1981 tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt
     2041 caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc
     2101 ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa
     2161 tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag
     2221 ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa
     2281 acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg
     2341 tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc
     2401 caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca
     2461 aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg
     2521 tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa
     2581 agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac
     2641 aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa
     2701 agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga
     2761 aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga
     2821 tattaatcgt ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga
     2881 cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa
     2941 cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc
     3001 caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag
     3061 tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa
     3121 gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact
     3181 tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga
     3241 tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct
     3301 aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt
     3361 ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat
     3421 aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga
     3481 aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac
     3541 tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat
     3601 gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc
     3661 aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa
     3721 aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt
     3781 ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat
     3841 ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga
     3901 agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg
     3961 tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc
     4021 aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc
     4081 agaagataac gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat
     4141 tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa
     4201 agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat
     4261 tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac
     4321 aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca
     4381 tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacta
     4441 atcgctggga cgcccgccat ggttcagcca aaaaacttaa gaccgccggt cttgtccact
     4501 accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc aattcttctg
     4561 acctgtaacg aataatagat agtaaagtag tctccgattg agttttctct gccgagtccc
     4621 acccagttct gtgatttcag taagttggta attgatacac tgttgcgaga actgctgcct
     4681 ggtagtagat aggttgttat tgagtaagaa ggtaaagtga acgaaatccc tgaaactgag
     4741 actgtagaaa ataagcttgt ccagactatt cctgcaggct cggtaccaaa ttccagaaaa
     4801 gaggcctccc gaaagggggg ccttttttcg ttttggtcct actgattggg tgaactacga
     4861 tacttccaga ataacgagaa atacgctact cttacagata aaactcactg ccgattacgg
     4921 ataggacgaa caacactttg attatcttac gatttaccga aggttctatt gagttggacc
     4981 cagcagttac tacttttatt cagtgattct gtcttatcgg gagattgttc gcttaccacg
     5041 tcgaccagcc tgcggtccgg tttacggcta gctcagtcct aggtattatg ctagctcgct
     5101 gggacgcccg agatagccgt tacacaggtg acacttattt cagcctgcgg tccggggtac
     5161 cattaaagag gagaaagaat tcatggacca ctacctcgac attcgcttgc gaccggaccc
     5221 ggaatttccc ccggcgcaac tcatgagcgt gctcttcggc aagctccacc aggccctggt
     5281 ggcacagggc ggggacagga tcggcgtgag cttccccgac ctcgacgaaa gccgctcccg
     5341 gctgggcgag cgcctgcgca ttcatgcctc ggcggacgac cttcgtgccc tgctcgcccg
     5401 gccctggctg gaagggttgc gggaccatct gcaattcgga gaaccggcag tcgtgcctca
     5461 ccccacaccg taccgtcagg tcagtcgggt tcaggcgaaa agcaatccgg aacgcctgcg
     5521 gcggcggctc atgcgccggc acgatctgag tgaggaggag gctcggaaac gcattcccga
     5581 tacggtcgcg agagccttgg acctgccctt cgtcacgcta cgcagccaga gcaccggaca
     5641 gcacttccgt ctcttcatcc gccacgggcc gttgcaggtg acggcagagg aaggaggatt
     5701 cacctgttac gggttgagca aaggaggttt cgttccctgg ttctgataat cgctgggacg
     5761 cccggcggcc gcggaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc
     5821 gaccaaaggt agcgaacgac gagtcactgt tgaggataaa tactttctct actaggcgcc
     5881 tgttacacag gtcctcagcg gcgcgcctgc tgccaccgct gagcaataac tagcataacc
     5941 ccttggggcc tctaaacggg tcttgagggg ttttttgcca ggcatcaaat aaaacgaaag
     6001 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     6061 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     6121 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     6181 gatggccttt ttgcgtttca gatctaccgg taaaccagca atagacataa gcggctattt
     6241 aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc
     6301 ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
     6361 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
     6421 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
     6481 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
     6541 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
     6601 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
     6661 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
     6721 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     6781 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     6841 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     6901 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     6961 gcaaatcaat atcactgtgt ggcttcaggc cgtcatccac tgcggagccg tacaaatgta
     7021 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     7081 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     7141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7201 aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga
     7261 cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa
     7321 cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat
     7381 aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag
     7441 tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct
     7501 tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac
     7561 gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt
     7621 tgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc
     7681 ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc
     7741 attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt
     7801 gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc
     7861 agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag
     7921 gtggtttttt cgaaggttaa gtcagttggg gaactgctta accttcgatc aaaccacctc
     7981 cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa ggatctcaag
     8041 aagatccttt gatc
//
