LOCUS       sequence-409070-        7095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  J23113.dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7095)
  AUTHORS   Alba Burbano D, Cardiff RAL, Tickman BI, Kiattisewee C, Maranas CJ, 
            Zalatan JG, Carothers JM
  TITLE     Engineering activatable promoters for scalable and multi-input 
            CRISPRa/i circuits.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jul 25;120(30):e2220358120. doi: 
            10.1073/pnas.2220358120. Epub 2023 Jul 18.
  PUBMED    37463216
REFERENCE   2  (bases 1 to 7095)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-07-25"; volume: "120(30):e2220358120. 
            doi"; pages: " 10.1073/pnas.2220358120. Epub 2023 Jul 18"
COMMENT     Sequence Label: pDA010.188
FEATURES             Location/Qualifiers
     source          1..7095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(448..1239,1240..1308))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1071..1090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1309..1413)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(1914..1957)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1966..2000
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     RBS             2010..2021
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2028..2054,2055..2057,2058..4544,4545..4547,
                     4548..6134)
                     /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"
     primer_bind     complement(6613..6630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6784..7095,1..277))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6864..6883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
       61 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
      121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
      181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
      241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
      301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
      361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
      421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
      481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
      541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
      601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
      661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
      721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
      781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
      841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
      901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
      961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     1021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     1081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     1141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     1201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     1261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     1321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     1381 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     1441 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     1501 ctttcgtctt caagaattct ggcgaatcct ctgaccagcc agaaaacgac ctttctgtgg
     1561 tgaaaccgga tgctgcaatt cagagcgcca gcaagtgggg gacagcagaa gacctgaccg
     1621 ccgcagagtg gatgtttgac atggtgaaga ctatcgcacc atcagccaga aaaccgaatt
     1681 ttgctgggtg ggctaacgat atccgcctga tgcgtgaacg tgacggacgt aaccaccgcg
     1741 acatgtgtgt gctgttccgc tgggcatgcc aggacaactt ctggtccggt aacgtgctga
     1801 gcccggccaa gcttagcccg aaaagtgcca cctgtggcaa ttccgacgtc ccgggtaaga
     1861 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag
     1921 ataaaaaaaa tccttagctt tcgctaagga tgatttctga tgcatctgat ggctagctca
     1981 gtcctaggga ttatgctagc gaattcatta aagaggagaa aatgcatatg gataagaaat
     2041 actcaatagg cttagctatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat
     2101 ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa
     2161 aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca
     2221 aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga
     2281 ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt
     2341 ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg
     2401 aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt
     2461 ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc
     2521 gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat
     2581 ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg
     2641 gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc
     2701 tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc attgctttgt
     2761 cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac
     2821 agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa attggagatc
     2881 aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata
     2941 tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct
     3001 acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag
     3061 aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg
     3121 ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg
     3181 gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct
     3241 ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa
     3301 gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga
     3361 cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga
     3421 tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag
     3481 gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg
     3541 aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga
     3601 caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga
     3661 agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa
     3721 aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag
     3781 atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag
     3841 attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct
     3901 tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg
     3961 ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa
     4021 aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat
     4081 cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta
     4141 aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg
     4201 caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg
     4261 atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac
     4321 gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg
     4381 aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc
     4441 aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg
     4501 accaagaatt agatattaat cgtttaagtg attatgatgt cgatgccatt gttccacaaa
     4561 gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg
     4621 gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac
     4681 aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac
     4741 gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc
     4801 gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg
     4861 aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg
     4921 acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc
     4981 atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg
     5041 aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt
     5101 ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact
     5161 tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa
     5221 ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca
     5281 aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat
     5341 tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag
     5401 actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag
     5461 tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag
     5521 ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta
     5581 aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg
     5641 agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg
     5701 agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt
     5761 tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt
     5821 atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg
     5881 ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac
     5941 aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta
     6001 aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg
     6061 ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc
     6121 taggaggtga ctaacaacgc atgagaaagc ccccggaaga tcaccttccg ggggcttttt
     6181 tattgcgcgg atccttactc cgtcgacaag cttgcggccg cactcgagga attccgactc
     6241 aattagttca gtcagtttca ggatattagt catctctaca ttgattatga gtattcagaa
     6301 attccttaaa tattctgaca aatgctcttt ccctaaactc cccccataaa aaaacccgcc
     6361 gaagcgggtt tttacgttat ttgcggatta acgattactc gttatcagaa ccgcccagac
     6421 ctgcgttcag cagttctgcc aggctggcag atgcgtcttc cggaattgat ccgtcgaccg
     6481 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     6541 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     6601 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6661 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6721 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6781 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6841 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6901 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6961 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7021 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7081 taactatcgt cttga
//
