LOCUS       sequence-488019-        8964 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of dCas9, alpha-NTD-MCP, and 
            PspF-lambda-N22.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8964)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 8964)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK761
FEATURES             Location/Qualifiers
     source          1..8964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(113..207)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(228..887)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     802..821
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(888..990)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(1194..1272)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(1568..1594,1595..1597,1598..4084,4085..4087,
                     4088..5674)
                     /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"
     terminator      5698..5769
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5785..5812
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        5869..5903
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             5913..5924
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             8201..8212
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(8222..8256)
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     rep_origin      complement(8418..8963)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(8498..8517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa
       61 atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgctt ggattctcac
      121 caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg
      181 tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta cgccccgccc
      241 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
      301 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
      361 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
      421 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
      481 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
      541 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
      601 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
      661 gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
      721 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
      781 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
      841 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
      901 gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
      961 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat atcgacgtct
     1021 taagttacga aatcatcctg tggagcttag taggtttagc aagatggcag cgcctaaatg
     1081 tagaatgata aaaggattaa gagattaatt tccctaaaaa tgataaaaca agcgttttga
     1141 aagcgcttgt ttttttggtt tgcagtcaga gtagaataga agtatcaaaa aaagcaccga
     1201 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatgctgtt
     1261 ttgaatggtt ccaacaagat tattttataa cttttataac aaataatcaa ggagaaattc
     1321 aaagaaattt atcagccata aaacaatact taatactata gaatgataac aaaataaact
     1381 actttttaaa agaattttgt gttataatct atttattatt aagtattggg taatattttt
     1441 tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg gaggtcatta
     1501 aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt tattttagga
     1561 ggcaaaaatg gataagaaat actcaatagg cttagctatc ggcacaaata gcgtcggatg
     1621 ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac
     1681 agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac
     1741 agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg
     1801 tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt
     1861 tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat
     1921 ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct
     1981 gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt
     2041 agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa
     2101 tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga
     2161 aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa
     2221 atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt
     2281 tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt
     2341 ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt tagataattt
     2401 attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga
     2461 tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc
     2521 agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt
     2581 agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg
     2641 atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc
     2701 aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt
     2761 gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga
     2821 gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga
     2881 gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg
     2941 caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt
     3001 tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt
     3061 tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt
     3121 tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc
     3181 atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa
     3241 agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt
     3301 tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct
     3361 aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga
     3421 tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac
     3481 atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg
     3541 ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac
     3601 aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc agctgatcca
     3661 tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga
     3721 tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt
     3781 acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa
     3841 tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg
     3901 agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga
     3961 gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa
     4021 tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt
     4081 cgatgccatt gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac
     4141 gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa
     4201 gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga
     4261 taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa
     4321 acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg
     4381 catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt
     4441 aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat
     4501 taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat
     4561 taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt
     4621 tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt
     4681 ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg
     4741 caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg
     4801 agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac
     4861 agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa
     4921 gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac
     4981 ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa
     5041 atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc
     5101 gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact
     5161 acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg
     5221 agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt
     5281 agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt
     5341 tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg aattttctaa
     5401 gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag
     5461 agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct
     5521 tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc
     5581 tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac
     5641 acgcattgat ttgagtcagc taggaggtga ctaactcgag taaggatctc caggcatcaa
     5701 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     5761 aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacc
     5821 tagagcgccc ggaagagagt caattcaggg tggtgaatcc ccagatcttt tacggctagc
     5881 tcagccctag gtattatgct agcgaattca ttaaagagga gaaaggtacc atggggccca
     5941 tgcagggttc tgtgacagag tttctaaaac cgcgcctggt tgatatcgag caagtgagtt
     6001 cgacgcacgc caaggtgacc cttgagcctt tagagcgtgg ctttggccat actctgggta
     6061 acgcactgcg ccgtattctg ctctcatcga tgccgggttg cgcggtgacc gaggttgaga
     6121 ttgatggtgt actacatgag tacagcacca aagaaggcgt tcaggaagat atcctggaaa
     6181 tcctgctcaa cctgaaaggg ctggcggtga gagttcaggg caaagatgaa gttattctta
     6241 ccttgaataa atctggcatt ggccctgtga ctgcagccga tatcacccac gacggtgatg
     6301 tcgaaatcgt caagccgcag cacgtgatct gccacctgac cgatgagaac gcgtctatta
     6361 gcatgcgtat caaagttcag cgcggtcgtg gttatgtgcc ggcttctacc cgaattcatt
     6421 cggaagaaga tgagcgccca atcggccgtc tgctggtcga cgcatgctac agccctgtgg
     6481 agcgtattgc ctacaatgtt gaagcagcgc gtgtagaaca gcgtaccgac ctggacaagc
     6541 tggtcatcga aatggaaacc aacggcacaa tcgatcctga agaggcgatt cgtcgtgcgg
     6601 caaccattct ggctgaacaa ctggaagctt tcgttgactt acgtgatgta cgtcagcctg
     6661 aagtgaaaga agagaaacca gaggcttcta actttactca gttcgttctc gtcgacaatg
     6721 gcggaactgg cgacgtgact gtcgccccaa gcaacttcgc taacgggatc gctgaatgga
     6781 tcagctctaa ctcgcgttca caggcttaca aagtaacctg tagcgttcgt cagagctctg
     6841 cgcagaatcg caaatacacc atcaaagtcg aggtgcctaa aggcgcctgg cgttcgtact
     6901 taaatatgga actaaccatt ccaattttcg ccacgaattc cgactgcgag cttattgtta
     6961 aggcaatgca aggtctccta aaagatggaa acccgattcc ctcagcaatc gcagcaaact
     7021 ccggcatcta ctaagcggcc gccacgcaaa aaaccccgct tcggcggggt tttttcgccc
     7081 tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     7141 gagcggaaat ggcttacgaa cggggcggag atttcctgcc cggggcgaaa aaaccccgcc
     7201 gaagcggggt tttttgcgtg ggcgcgcctt aatttgctgc tttccattga gcctgtttct
     7261 ctgcgcgacg ttcgcggcgg cgtgtgcgtg cattcatttg caaactgagt tgcagcaact
     7321 ctttttcctg ctgcatctga aactcacgta aatccagcgg cagtgttgga agcgaggtgg
     7381 tttctgaaac ggcgatagcg tcttcaggcg gacgccgttt aaagggatca ataatgatgt
     7441 catcaagcgg ataatcgctg gtgccgtggc gatacactga acgttccacc acgtttttca
     7501 attcacgaat atttcccggc caacgataat tcagcaatgt ttctctggcg cgctccgtaa
     7561 accccgggaa cagaggcagc ttgatttccc gacacatctg gatggcaaag tattctgcca
     7621 tcaacattat gtcgctttcg cgctcgcgca gtggtggcag ttgtacaaca tcaaaagcca
     7681 gtcggtcgag caggtcagcg cgaaaagtgc cttcattgac catcgccggg agatcggcat
     7741 tcgtcgcgca taccaaccgc acattcacct gcaatggttg gctgccgcca acgcgctcca
     7801 gttcaccgta ctcaatcacg cgcaataatt tctcctgcac catcatcggt gccgtagcga
     7861 gttcatcaag aaatagcgta ccgccgtcgg cacgttcaaa tctccctgga tgacgttttt
     7921 gcgcaccggt aaacgccccc gcttcgtgac caaacagttc ggaatccagc agattttcat
     7981 ttaacgccgc gcagttaagg gaaataaacg gcccttgcca acgggaggag agataatgca
     8041 ggcggctggc aatcagctct ttaccggtgc cgcgttcgcc gatgatgagc accggtttgt
     8101 ccagcggtgc gagatgcgaa acctgttcca gcacttcgag aaagctgttc gcctcaccaa
     8161 gtaaattatc tttgtattct gccatgggcc ccatggtacc tttctcctct ttaatgaatt
     8221 cgctagcata atacctaggg ctgagctagc cgtaaaatgc atcctaggga tatattccgc
     8281 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
     8341 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
     8401 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
     8461 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     8521 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
     8581 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
     8641 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
     8701 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
     8761 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
     8821 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
     8881 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
     8941 cgcgcagacc aaaacgatct caag
//
