LOCUS       sequence-488018-        8649 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of dCas9, MCP-TetD, and PspF-lambda-N22.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8649)
  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 8649)
  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: pCK948
FEATURES             Location/Qualifiers
     source          1..8649
                     /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             7886..7897
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(7907..7941)
                     /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(8103..8648)
                     /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(8183..8202)
                     /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 atggggcccg
     5941 cttctaactt tactcagttc gttctcgtcg acaatggcgg aactggcgac gtgactgtcg
     6001 ccccaagcaa cttcgctaac gggatcgctg aatggatcag ctctaactcg cgttcacagg
     6061 cttacaaagt aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa tacaccatca
     6121 aagtcgaggt gcctaaaggc gcctggcgtt cgtacttaaa tatggaacta accattccaa
     6181 ttttcgccac gaattccgac tgcgagctta ttgttaaggc aatgcaaggt ctcctaaaag
     6241 atggaaaccc gattccctca gcaatcgcag caaactccgg catctacggt ggcggaggta
     6301 gcatgtatat tgaacagcat tctcgctatc aaaataaagc taataacatc caattaagat
     6361 atgatgataa gcagtttcat acaacggtta tcaaagatgt tctattatgg attgaacata
     6421 atttagatca gtctttactg cttgatgatg tggcgaataa agcgggttat accaagtggt
     6481 attttcagcg gctgttcaaa aaagtaacag gggtcacact ggctagctat attcgtgctc
     6541 gtcgtttgac gaaagcggct gttgagttga ggttgacgaa aaaaactatc cttgagatcg
     6601 cattaaaata tcaatttgat tcccaacaat cttttacacg tcgatttaag tacattttta
     6661 aggttacacc aagttattat cggcgtaata aattatggga attggaggca atgcactaag
     6721 cggccgccac gcaaaaaacc ccgcttcggc ggggtttttt cgccctaggg atatattccg
     6781 cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt
     6841 acgaacgggg cggagatttc ctgcccgggg cgaaaaaacc ccgccgaagc ggggtttttt
     6901 gcgtgggcgc gccttaattt gctgctttcc attgagcctg tttctctgcg cgacgttcgc
     6961 ggcggcgtgt gcgtgcattc atttgcaaac tgagttgcag caactctttt tcctgctgca
     7021 tctgaaactc acgtaaatcc agcggcagtg ttggaagcga ggtggtttct gaaacggcga
     7081 tagcgtcttc aggcggacgc cgtttaaagg gatcaataat gatgtcatca agcggataat
     7141 cgctggtgcc gtggcgatac actgaacgtt ccaccacgtt tttcaattca cgaatatttc
     7201 ccggccaacg ataattcagc aatgtttctc tggcgcgctc cgtaaacccc gggaacagag
     7261 gcagcttgat ttcccgacac atctggatgg caaagtattc tgccatcaac attatgtcgc
     7321 tttcgcgctc gcgcagtggt ggcagttgta caacatcaaa agccagtcgg tcgagcaggt
     7381 cagcgcgaaa agtgccttca ttgaccatcg ccgggagatc ggcattcgtc gcgcatacca
     7441 accgcacatt cacctgcaat ggttggctgc cgccaacgcg ctccagttca ccgtactcaa
     7501 tcacgcgcaa taatttctcc tgcaccatca tcggtgccgt agcgagttca tcaagaaata
     7561 gcgtaccgcc gtcggcacgt tcaaatctcc ctggatgacg tttttgcgca ccggtaaacg
     7621 cccccgcttc gtgaccaaac agttcggaat ccagcagatt ttcatttaac gccgcgcagt
     7681 taagggaaat aaacggccct tgccaacggg aggagagata atgcaggcgg ctggcaatca
     7741 gctctttacc ggtgccgcgt tcgccgatga tgagcaccgg tttgtccagc ggtgcgagat
     7801 gcgaaacctg ttccagcact tcgagaaagc tgttcgcctc accaagtaaa ttatctttgt
     7861 attctgccat gggccccatg gtacctttct cctctttaat gaattcgcta gcataatacc
     7921 tagggctgag ctagccgtaa aatgcatcct agggatatat tccgcttcct cgctcactga
     7981 ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga
     8041 tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg
     8101 tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg
     8161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg
     8221 cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg
     8281 tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat
     8341 gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     8401 caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg
     8461 agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact
     8521 gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa
     8581 aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac
     8641 gatctcaag
//
