LOCUS       sequence-459805-        8384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This YES-gate plasmid expresses dCas9 from a constitutive promoter, 
            GFP from a promoter repressible by sgRNA-Y, and sgRNA-Y repressible 
            by sgRNA-2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8384)
  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 8384)
  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_YES_i2
FEATURES             Location/Qualifiers
     source          1..8384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        98..173
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        221..255
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             318..326
                     /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             332..1045
                     /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(1249..1275,1276..1278,1279..3765,3766..3768,
                     3769..5355)
                     /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    5359..5379
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5434..5528
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5764..6555
                     /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            6701..6809
                     /note="incP origin of transfer"
     CDS             complement(7000..7950)
                     /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      7998..8220
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(join(8321..8384,1..23))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gactgagcct ttcgttttat ttgatgcctt taattaatag ctttacaccc ttgagctgct
       61 atggtattat gctagctatt tccgtgcctg ggtatgagtt ttagagctag aaatagcaag
      121 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
      181 tacgcagtaa gtctgtagag gcgatttccc aaccagtgct ttgacagcta gctcagtcct
      241 aggtataatg ctagctccat catacccagg cacggaaata tggcggcgcg attatccgcg
      301 ccttctctag gactttttaa ggaggtattt catgcgtaaa ggcgaagaac tgtttaccgg
      361 tgtggttccg attctggtgg aactggacgg cgatgttaat ggtcataaat tcagtgttcg
      421 cggcgaaggt gaaggcgatg cgacgaacgg caaactgacc ctgaaattta tctgcaccac
      481 gggtaaactg ccggtcccgt ggccgacgct ggtgaccacg ctgacctatg gcgttcaatg
      541 ttttgcgcgt tacccggatc acatgaaaca gcacgacttt ttcaaatcgg ccatgccgga
      601 aggctatgtg caggaacgta cgattagctt taaagacgat ggtacgtata aaacccgcgc
      661 ggaagtgaaa ttcgaaggcg ataccctggt taaccgtatc gaactgaaag gtatcgattt
      721 caaagaagac ggcaatattc tgggtcataa actggaatat aacttcaatt cccacaacgt
      781 gtacatcacc gcggataaac agaaaaacgg cattaaagcc aatttcaaaa tccgccataa
      841 tgtggaagat ggtagcgttc agctggccga ccactatcag caaaacacgc cgattggtga
      901 tggcccggtc ctgctgccgg acaatcacta cctgagtacc cagtccgtgc tgtcaaaaga
      961 tccgaacgaa aaacgtgacc acatggtcct gctggaattt gtgacggctg cgggtatcac
     1021 ccacggcatg gacgaactgt ataaaaggcc ttaataatac tcgaacccct agcccgctct
     1081 tatcgggcgg ctaggggttt tttgtcctag agacgaacaa taaggcctcc caaatcgggg
     1141 ggcctttttt attgataaca aaacctaggc caggcatcaa ttgaaatcat caaactcatt
     1201 atggatttaa tttaaactca agggacatca ggaggaggga attatagaat ggataagaaa
     1261 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
     1321 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
     1381 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
     1441 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
     1501 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
     1561 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
     1621 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
     1681 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
     1741 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
     1801 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1861 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1921 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1981 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     2041 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     2101 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     2161 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     2221 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     2281 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     2341 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     2401 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     2461 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     2521 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     2581 acttttcgta ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     2641 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     2701 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     2761 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     2821 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2881 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2941 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     3001 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     3061 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     3121 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     3181 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     3241 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     3301 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     3361 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     3421 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     3481 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     3541 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     3601 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     3661 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     3721 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
     3781 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3841 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3901 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3961 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     4021 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     4081 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     4141 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     4201 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     4261 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     4321 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     4381 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     4441 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     4501 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     4561 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     4621 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     4681 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     4741 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     4801 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4861 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4921 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4981 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     5041 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     5101 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     5161 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     5221 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     5281 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     5341 ctaggaggtg actgataata ctagtagcgg ccgctgcagg catgcaagct tgcggccgcg
     5401 tcgtgactgg gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga
     5461 cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg
     5521 gtgagaatcc aggggtcccc aataattacg atttacgtat ttaaatgaac cttgaccgaa
     5581 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     5641 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     5701 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     5761 atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     5821 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     5881 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     5941 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     6001 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     6061 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     6121 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     6181 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     6241 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     6301 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     6361 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     6421 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     6481 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     6541 gtagtcggca aataagacaa ttgtcctttt ccgctgcata accctgcttc ggggtcatta
     6601 tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg
     6661 ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
     6721 gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca
     6781 acgggaatcc tgctctgcga ggctggccgt aggccggcct ggtgaacagc tttgaatgca
     6841 ccaaaaactc gtaaaagctc tgatgtatct atctttttta caccgttttc atctgtgcat
     6901 atggacagtt ttccctttga tatgtaacgg tgaacagttg ttctactttt gtttgttagt
     6961 cttgatgctt cactgataga tacaagagcc ataagaacct cagatccttc cgtatttagc
     7021 cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag
     7081 atcatactta ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt
     7141 tgctgttaaa gcatcgtgta gtgtttttct tagtccgtta tgtaggtagg aatctgatgt
     7201 aatggttgtt ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac
     7261 gagatccatt tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg
     7321 cttatcaacc accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa
     7381 aacccattgg ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt
     7441 aaattcatca aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt
     7501 taataaccac tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag
     7561 attatatttt atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac
     7621 taattctaat ttttcgcttg agaacttggc atagtttgtc cactggaaga tcttaaagcc
     7681 tttaaccaaa ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttagc
     7741 taatacacca taagcatttt ccctactgat gttcatcatc tgagcgtatt ggttataagt
     7801 gaacgatacc gtccgttctt tccttgtagg gttttcaatc gtggggttga gtagtgccac
     7861 acagcataaa attagcttgg tttcatgctc cgttaagtca tagcgactaa tcgctagttc
     7921 atttgctttg aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc
     7981 actataccaa ttgagatggg ctagtcaatg ataattacta gtccttttcc tttgagttgt
     8041 gggtatctgt aaattctgct agacctttgc tggaaaactt gtaaattctg ctagaccctc
     8101 tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat attcaagtgg ttataattta
     8161 tagaataaag aaagaataaa aaaagataaa aagaatagat cccagccctg tgtataactc
     8221 actactttag tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct
     8281 acaaaacaga cctggcgcgc ccagctgtct agggcggcgg atttgtccta ctcaggagag
     8341 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttc
//
