LOCUS       sequence-459804-        8201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This NOT-gate plasmid expresses dCas9 from a constitutive promoter, 
            and GFP from a promoter repressible by sgRNA-1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8201)
  AUTHORS   Pujar A, Pathania A, Hopper C, Pandi A, Calderon CR, Fugger M, Nowak
            T, Kushwaha M
  TITLE     Phage-mediated intercellular CRISPRi for biocomputation in bacterial
            consortia.
  JOURNAL   Nucleic Acids Res. 2024 Dec 27:gkae1256. doi: 10.1093/nar/gkae1256.
  PUBMED    39727169
REFERENCE   2  (bases 1 to 8201)
  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: "Nucleic 
            Acids Res. 2024 Dec 27:gkae1256. doi: 10.1093/nar/gkae1256."
COMMENT     Sequence Label: pSEVA47_dCas9-GFP_NOT_i1
FEATURES             Location/Qualifiers
     source          1..8201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..74
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             133..141
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             147..860
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1064..1090,1091..1093,1094..3580,3581..3583,
                     3584..5170)
                     /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"
     misc_feature    5174..5194
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5249..5343
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5579..6370
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     oriT            6516..6624
                     /note="incP origin of transfer"
     CDS             complement(6815..7765)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFKIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      7813..8035
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(join(8136..8201,1..21))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgagccttt cgttttattt gatgccttta attaatagct tgacagctag ctcagtccta
       61 ggtataatgc tagctatttc cgtgcctggg tatgatggcg gcgcgattat ccgcgccttc
      121 tctaggactt tttaaggagg tatttcatgc gtaaaggcga agaactgttt accggtgtgg
      181 ttccgattct ggtggaactg gacggcgatg ttaatggtca taaattcagt gttcgcggcg
      241 aaggtgaagg cgatgcgacg aacggcaaac tgaccctgaa atttatctgc accacgggta
      301 aactgccggt cccgtggccg acgctggtga ccacgctgac ctatggcgtt caatgttttg
      361 cgcgttaccc ggatcacatg aaacagcacg actttttcaa atcggccatg ccggaaggct
      421 atgtgcagga acgtacgatt agctttaaag acgatggtac gtataaaacc cgcgcggaag
      481 tgaaattcga aggcgatacc ctggttaacc gtatcgaact gaaaggtatc gatttcaaag
      541 aagacggcaa tattctgggt cataaactgg aatataactt caattcccac aacgtgtaca
      601 tcaccgcgga taaacagaaa aacggcatta aagccaattt caaaatccgc cataatgtgg
      661 aagatggtag cgttcagctg gccgaccact atcagcaaaa cacgccgatt ggtgatggcc
      721 cggtcctgct gccggacaat cactacctga gtacccagtc cgtgctgtca aaagatccga
      781 acgaaaaacg tgaccacatg gtcctgctgg aatttgtgac ggctgcgggt atcacccacg
      841 gcatggacga actgtataaa aggccttaat aatactcgaa cccctagccc gctcttatcg
      901 ggcggctagg ggttttttgt cctagagacg aacaataagg cctcccaaat cggggggcct
      961 tttttattga taacaaaacc taggccaggc atcaattgaa atcatcaaac tcattatgga
     1021 tttaatttaa actcaaggga catcaggagg agggaattat agaatggata agaaatactc
     1081 aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg atgaatataa
     1141 ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta tcaaaaaaaa
     1201 tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc gtctcaaacg
     1261 gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac aggagatttt
     1321 ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag agtctttttt
     1381 ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag tagatgaagt
     1441 tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg tagattctac
     1501 tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta agtttcgtgg
     1561 tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca aactatttat
     1621 ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg caagtggagt
     1681 agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag aaaatctcat
     1741 tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg ctttgtcatt
     1801 gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta aattacagct
     1861 ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg gagatcaata
     1921 tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt cagatatcct
     1981 aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta aacgctacga
     2041 tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac ttccagaaaa
     2101 gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata ttgatggggg
     2161 agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa tggatggtac
     2221 tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac ggacctttga
     2281 caacggctct attccccatc aaattcactt gggtgagctg catgctattt tgagaagaca
     2341 agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt
     2401 tcgtattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg catggatgac
     2461 tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc
     2521 ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc caaatgaaaa
     2581 agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg aattgacaaa
     2641 ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg aacagaagaa
     2701 agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga
     2761 agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag ttgaagatag
     2821 atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag ataaagattt
     2881 tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat tgaccttatt
     2941 tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct ttgatgataa
     3001 ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt
     3061 gattaatggt attagggata agcaatctgg caaaacaata ttagattttt tgaaatcaga
     3121 tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga catttaaaga
     3181 agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac atattgcaaa
     3241 tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag ttgttgatga
     3301 attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa tggcacgtga
     3361 aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga
     3421 aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa atactcaatt
     3481 gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt atgtggacca
     3541 agaattagat attaatcgtt taagtgatta tgatgtcgat gccattgttc cacaaagttt
     3601 ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa atcgtggtaa
     3661 atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt ggagacaact
     3721 tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg
     3781 aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg aaactcgcca
     3841 aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat acgatgaaaa
     3901 tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag tttctgactt
     3961 ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc atgcccatga
     4021 tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa aacttgaatc
     4081 ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg ctaagtctga
     4141 gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca tgaacttctt
     4201 caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa tcgaaactaa
     4261 tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag tgcgcaaagt
     4321 attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag gcggattctc
     4381 caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta aaaaagactg
     4441 ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag tcctagtggt
     4501 tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt tactagggat
     4561 cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag aagctaaagg
     4621 atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc tttttgagtt
     4681 agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag gaaatgagct
     4741 ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg aaaagttgaa
     4801 gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata agcattattt
     4861 agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag cagatgccaa
     4921 tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac gtgaacaagc
     4981 agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg cttttaaata
     5041 ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt tagatgccac
     5101 tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga gtcagctagg
     5161 aggtgactga taatactagt agcggccgct gcaggcatgc aagcttgcgg ccgcgtcgtg
     5221 actgggaaaa ccctggcgac tagtcttgga ctcctgttga tagatccagt aatgacctca
     5281 gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag
     5341 aatccagggg tccccaataa ttacgattta cgtatttaaa tgaaccttga ccgaacgcag
     5401 cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca
     5461 gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt
     5521 atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacatcat
     5581 gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga
     5641 gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg
     5701 cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac
     5761 aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga
     5821 gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta
     5881 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat
     5941 cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca
     6001 tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga
     6061 tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg
     6121 cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa
     6181 aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca
     6241 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc
     6301 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt
     6361 cggcaaataa gacaattgtc cttttccgct gcataaccct gcttcggggt cattatagcg
     6421 attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt
     6481 gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca
     6541 cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg
     6601 aatcctgctc tgcgaggctg gccgtaggcc ggcctggtga acagctttga atgcaccaaa
     6661 aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga
     6721 cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga
     6781 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     6841 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     6901 acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgctg
     6961 ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg
     7021 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     7081 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     7141 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     7201 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     7261 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     7321 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     7381 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     7441 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaagatctta aagcctttaa
     7501 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     7561 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
     7621 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     7681 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     7741 ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt taatcactat
     7801 accaattgag atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta
     7861 tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa
     7921 attccgctag acctttgtgt gttttttttg tttatattca agtggttata atttatagaa
     7981 taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac
     8041 tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa
     8101 acagacctgg cgcgcccagc tgtctagggc ggcggatttg tcctactcag gagagcgttc
     8161 accgacaaac aacagataaa acgaaaggcc cagtctttcg a
//
