LOCUS       sequence-489267-        8646 bp    DNA     circular SYN 14-OCT-2025
DEFINITION  Constitutive expression of dCas9, MCP-SoxS-SYNZIP6, and 
            alpha-NTD-SYNZIP5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8646)
  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 8646)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 14, 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: pCK883
FEATURES             Location/Qualifiers
     source          1..8646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(114..208)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(229..888)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     803..822
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(889..991)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(1195..1273)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(1569..1595,1596..1598,1599..4085,4086..4088,
                     4089..5675)
                     /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      5699..5770
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5786..5813
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        5870..5904
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             5914..5925
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             7884..7895
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(7905..7939)
                     /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(8101..8646)
                     /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(8181..8200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca
       61 aatgtagcac ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca
      121 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
      181 gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt acgccccgcc
      241 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
      301 acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
      361 atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
      421 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
      481 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
      541 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
      601 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
      661 tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
      721 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
      781 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
      841 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
      901 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
      961 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaga tatcgacgtc
     1021 ttaagttacg aaatcatcct gtggagctta gtaggtttag caagatggca gcgcctaaat
     1081 gtagaatgat aaaaggatta agagattaat ttccctaaaa atgataaaac aagcgttttg
     1141 aaagcgcttg tttttttggt ttgcagtcag agtagaatag aagtatcaaa aaaagcaccg
     1201 actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatgctgt
     1261 tttgaatggt tccaacaaga ttattttata acttttataa caaataatca aggagaaatt
     1321 caaagaaatt tatcagccat aaaacaatac ttaatactat agaatgataa caaaataaac
     1381 tactttttaa aagaattttg tgttataatc tatttattat taagtattgg gtaatatttt
     1441 ttgaagagat attttgaaaa agaaaaatta aagcatatta aactaatttc ggaggtcatt
     1501 aaaactatta ttgaaatcat caaactcatt atggatttaa tttaaacttt ttattttagg
     1561 aggcaaaaat ggataagaaa tactcaatag gcttagctat cggcacaaat agcgtcggat
     1621 gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata
     1681 cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac agtggagaga
     1741 cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt cggaagaatc
     1801 gtatttgtta tctacaggag attttttcaa atgagatggc gaaagtagat gatagtttct
     1861 ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa cgtcatccta
     1921 tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact atctatcatc
     1981 tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc tatttggcct
     2041 tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta aatcctgata
     2101 atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa ttatttgaag
     2161 aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca cgattgagta
     2221 aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa aatggcttat
     2281 ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca aattttgatt
     2341 tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat ttagataatt
     2401 tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag aatttatcag
     2461 atgctatttt actttcagat atcctaagag taaatactga aataactaag gctcccctat
     2521 cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt ttaaaagctt
     2581 tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg
     2641 gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa tttatcaaac
     2701 caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat cgtgaagatt
     2761 tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt cacttgggtg
     2821 agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa gacaatcgtg
     2881 agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca ttggcgcgtg
     2941 gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc ccatggaatt
     3001 ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc atgacaaact
     3061 ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt tatgagtatt
     3121 ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag
     3181 catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa
     3241 aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg
     3301 ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc
     3361 taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg
     3421 atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa
     3481 catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg
     3541 gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa
     3601 caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg cagctgatcc
     3661 atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg
     3721 atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa aaaggtattt
     3781 tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat aagccagaaa
     3841 atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag aaaaattcgc
     3901 gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag attcttaaag
     3961 agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat tatctccaaa
     4021 atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt gattatgatg
     4081 tcgatgccat tgttccacaa agtttcctta aagacgattc aatagacaat aaggtcttaa
     4141 cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa
     4201 agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa cgtaagtttg
     4261 ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct ggttttatca
     4321 aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc
     4381 gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa gtgattacct
     4441 taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa gtacgtgaga
     4501 ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga actgctttga
     4561 ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa gtttatgatg
     4621 ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca aaatatttct
     4681 tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat ggagagattc
     4741 gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg gataaagggc
     4801 gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa
     4861 cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga aattcggaca
     4921 agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt gatagtccaa
     4981 cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg aagaagttaa
     5041 aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt gaaaaaaatc
     5101 cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta atcattaaac
     5161 tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg gctagtgccg
     5221 gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat tttttatatt
     5281 tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa aaacaattgt
     5341 ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt gaattttcta
     5401 agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat aacaaacata
     5461 gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg ttgacgaatc
     5521 ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa cgatatacgt
     5581 ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt ctttatgaaa
     5641 cacgcattga tttgagtcag ctaggaggtg actaactcga gtaaggatct ccaggcatca
     5701 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     5761 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac
     5821 ctagagcgcc cggaagagag tcaattcagg gtggtgaatc cccagatctt ttacggctag
     5881 ctcagcccta ggtattatgc tagcgaattc attaaagagg agaaaggtac catggggccc
     5941 gcttctaact ttactcagtt cgttctcgtc gacaatggcg gaactggcga cgtgactgtc
     6001 gccccaagca acttcgctaa cgggatcgct gaatggatca gctctaactc gcgttcacag
     6061 gcttacaaag taacctgtag cgttcgtcag agctctgcgc agaatcgcaa atacaccatc
     6121 aaagtcgagg tgcctaaagg cgcctggcgt tcgtacttaa atatggaact aaccattcca
     6181 attttcgcca cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa
     6241 gatggaaacc cgattccctc agcaatcgca gcaaactccg gcatctacgg tggcggaggt
     6301 agcatgtccc atcagaaaat tattcaggat cttatcgcat ggattgacga gcatattgac
     6361 cagccgctta acattgatgt agtcgcaaaa aaatcaggct attcaaagtg gtacttgcaa
     6421 cgaatgttcc gcacggtgac gcatcagacg cttggcgatt acattcgcca acgccgcctg
     6481 ttactggccg ccgttgagtt gcgcaccacc gagcgtccga tttttgatat cgcaatggac
     6541 ctgggttatg tctcgcagca gaccttctcc cgcgttttcg cgcggcagtt tgatcgcact
     6601 cccgcggatt atcgccaccg cctgggtggc ggaggtagcc aaaaagttgc gcagctgaaa
     6661 aaccgtgttg cgtacaaact gaaagaaaac gcgaagctgg agaacatcgt ggcgcgtctg
     6721 gaaaacgaca atgcgaacct ggagaaagac attgcgaatc tcgaaaagga catcgcaaat
     6781 ctggaacgtg acgttgcgcg ttaagcggcc gccacgcaaa aaaccccgct tcggcggggt
     6841 tttttcgccc tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     6901 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgcc cggggcgaaa
     6961 aaaccccgcc gaagcggggt tttttgcgtg ggcgcgcctt actcgaattt gtgagccgcc
     7021 agttcgaatt ccagttccgc ttttgcgaat ttcaggtgtt ccttcaggtt tttgagttca
     7081 gcgttacgct cttccagctc ctggatgtag tttttcagtt ctttaacggt gttctctggt
     7141 ttctcttctt tcacttcagg ctgacgtaca tcacgtaagt caacgaaagc ttccagttgt
     7201 tcagccagaa tggttgccgc acgacgaatc gcctcttcag gatcgattgt gccgttggtt
     7261 tccatttcga tgaccagctt gtccaggtcg gtacgctgtt ctacacgcgc tgcttcaaca
     7321 ttgtaggcaa tacgctccac agggctgtag catgcgtcga ccagcagacg gccgattggg
     7381 cgctcatctt cttccgaatg aattcgggta gaagccggca cataaccacg accgcgctga
     7441 actttgatac gcatgctaat agacgcgttc tcatcggtca ggtggcagat cacgtgctgc
     7501 ggcttgacga tttcgacatc accgtcgtgg gtgatatcgg ctgcagtcac agggccaatg
     7561 ccagatttat tcaaggtaag aataacttca tctttgccct gaactctcac cgccagccct
     7621 ttcaggttga gcaggatttc caggatatct tcctgaacgc cttctttggt gctgtactca
     7681 tgtagtacac catcaatctc aacctcggtc accgcgcaac ccggcatcga tgagagcaga
     7741 atacggcgca gtgcgttacc cagagtatgg ccaaagccac gctctaaagg ctcaagggtc
     7801 accttggcgt gcgtcgaact cacttgctcg atatcaacca ggcgcggttt tagaaactct
     7861 gtcacagaac cctgcatggt acctttctcc tctttaatga attcgctagc ataataccta
     7921 gggctgagct agccgtaaaa tgcatcctag ggatatattc cgcttcctcg ctcactgact
     7981 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     8041 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     8101 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     8161 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg
     8221 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     8281 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     8341 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     8401 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     8461 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     8521 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     8581 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     8641 tctcaa
//
