LOCUS       sequence-461749-        8159 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-A1. (This plasmid is a 
            gift of the Jaramillo Lab, where it is called PAJ285.).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8159)
  AUTHORS   Kushwaha M, Singh V, Jaramillo A
  TITLE     Development of a genetically-encoded in vivo computing platform to 
            play the game of Tic-Tac-Toe
  JOURNAL   Zenodo (2017) Conference: SB7.0: The Seventh International Meeting 
            on Synthetic Biology.
REFERENCE   2  (bases 1 to 8159)
  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: "Zenodo 
            (2017) Conference: SB7.0: The Seventh International Meeting on 
            Synthetic Biology."
COMMENT     Sequence Label: pSEVA47_dCas9-GFP_NOT_iA
FEATURES             Location/Qualifiers
     source          1..8159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             77..790
                     /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(994..1020,1021..1023,1024..3510,3511..3513,3514..5100)
                     /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    5104..5124
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5179..5273
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5509..6300
                     /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            6446..6554
                     /note="incP origin of transfer"
     CDS             complement(6745..7695)
                     /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      7743..7965
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(8066..8152)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 taattaatag ctttacaccc tagcgtagct ttggtattat gctagcttct agacaattaa
       61 tagggaggtt ctcgagatgc gtaaaggcga agaactgttt accggtgtgg ttccgattct
      121 ggtggaactg gacggcgatg ttaatggtca taaattcagt gttcgcggcg aaggtgaagg
      181 cgatgcgacg aacggcaaac tgaccctgaa atttatctgc accacgggta aactgccggt
      241 cccgtggccg acgctggtga ccacgctgac ctatggcgtt caatgttttg cgcgttaccc
      301 ggatcacatg aaacagcacg actttttcaa atcggccatg ccggaaggct atgtgcagga
      361 acgtacgatt agctttaaag acgatggtac gtataaaacc cgcgcggaag tgaaattcga
      421 aggcgatacc ctggttaacc gtatcgaact gaaaggtatc gatttcaaag aagacggcaa
      481 tattctgggt cataaactgg aatataactt caattcccac aacgtgtaca tcaccgcgga
      541 taaacagaaa aacggcatta aagccaattt caaaatccgc cataatgtgg aagatggtag
      601 cgttcagctg gccgaccact atcagcaaaa cacgccgatt ggtgatggcc cggtcctgct
      661 gccggacaat cactacctga gtacccagtc cgtgctgtca aaagatccga acgaaaaacg
      721 tgaccacatg gtcctgctgg aatttgtgac ggctgcgggt atcacccacg gcatggacga
      781 actgtataaa aggccttaat aatactcgaa cccctagccc gctcttatcg ggcggctagg
      841 ggttttttgt cctagagacg aacaataagg cctcccaaat cggggggcct tttttattga
      901 taacaaaacc taggccaggc atcaattgaa atcatcaaac tcattatgga tttaatttaa
      961 actcaaggga catcaggagg agggaattat agaatggata agaaatactc aataggctta
     1021 gctatcggca caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct
     1081 aaaaagttca aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg
     1141 gctcttttat ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt
     1201 agaaggtata cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag
     1261 atggcgaaag tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa
     1321 gacaagaagc atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat
     1381 gagaaatatc caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg
     1441 gatttgcgct taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg
     1501 attgagggag atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta
     1561 caaacctaca atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa
     1621 gcgattcttt ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc
     1681 cccggtgaga agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc
     1741 cctaatttta aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat
     1801 acttacgatg atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg
     1861 tttttggcag ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat
     1921 actgaaataa ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat
     1981 caagacttga ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa
     2041 atcttttttg atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa
     2101 gaagaatttt ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta
     2161 ttggtgaaac taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct
     2221 attccccatc aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt
     2281 tatccatttt taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgtattcct
     2341 tattatgttg gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct
     2401 gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa
     2461 tcatttattg aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca
     2521 aaacatagtt tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat
     2581 gttactgaag gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt
     2641 gatttactct tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc
     2701 aaaaaaatag aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct
     2761 tcattaggta cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat
     2821 gaagaaaatg aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg
     2881 gagatgattg aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa
     2941 cagcttaaac gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt
     3001 attagggata agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc
     3061 aatcgcaatt ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa
     3121 aaagcacaag tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt
     3181 agccctgcta ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa
     3241 gtaatggggc ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca
     3301 actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa
     3361 gaattaggaa gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa
     3421 aagctctatc tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat
     3481 attaatcgtt taagtgatta tgatgtcgat gccattgttc cacaaagttt ccttaaagac
     3541 gattcaatag acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac
     3601 gttccaagtg aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc
     3661 aagttaatca ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt
     3721 gaacttgata aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag
     3781 catgtggcac aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt
     3841 attcgagagg ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat
     3901 ttccaattct ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta
     3961 aatgccgtcg ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc
     4021 tatggtgatt ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata
     4081 ggcaaagcaa ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa
     4141 attacacttg caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact
     4201 ggagaaattg tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg
     4261 ccccaagtca atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca
     4321 attttaccaa aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa
     4381 aaatatggtg gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg
     4441 gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg
     4501 gaaagaagtt cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa
     4561 gttaaaaaag acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt
     4621 cgtaaacgga tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca
     4681 agcaaatatg tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca
     4741 gaagataacg aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt
     4801 attgagcaaa tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa
     4861 gttcttagtg catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt
     4921 attcatttat ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca
     4981 acaattgatc gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat
     5041 caatccatca ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga
     5101 taatactagt agcggccgct gcaggcatgc aagcttgcgg ccgcgtcgtg actgggaaaa
     5161 ccctggcgac tagtcttgga ctcctgttga tagatccagt aatgacctca gaactccatc
     5221 tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccagggg
     5281 tccccaataa ttacgattta cgtatttaaa tgaaccttga ccgaacgcag cggtggtaac
     5341 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct
     5401 cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc
     5461 aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacatcat gagggaagcg
     5521 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
     5581 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
     5641 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     5701 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     5761 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     5821 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     5881 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     5941 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     6001 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     6061 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     6121 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     6181 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     6241 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     6301 gacaattgtc cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     6361 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     6421 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     6481 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     6541 tgcgaggctg gccgtaggcc ggcctggtga acagctttga atgcaccaaa aactcgtaaa
     6601 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc
     6661 tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg
     6721 atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag
     6781 tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg
     6841 catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgctg ttaaagcatc
     6901 gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat
     6961 tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct
     7021 atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa
     7081 tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag
     7141 ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct
     7201 aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata
     7261 aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa
     7321 tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc
     7381 gcttgagaac ttggcatagt ttgtccactg gaagatctta aagcctttaa ccaaaggatt
     7441 cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc
     7501 attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg
     7561 ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag
     7621 cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac
     7681 aactaattca gacatacatc tcaattggtc taggtgattt taatcactat accaattgag
     7741 atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt
     7801 ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag
     7861 acctttgtgt gttttttttg tttatattca agtggttata atttatagaa taaagaaaga
     7921 ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt
     7981 tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctgg
     8041 cgcgcccagc tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac
     8101 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctt
//
