LOCUS       sequence-486134-        7011 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  aTc-inducible expression of dCas9-AsiA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  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 7011)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 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: pCK320
FEATURES             Location/Qualifiers
     source          1..7011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..633)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(740..766,767..769,770..3256,3257..3259,3260..4843)
                     /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      5176..5247
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5263..5290
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5318..5335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5452..5997)
                     /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(5532..5551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6111..6205)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6226..6885)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6800..6819
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6886..6988)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taaagaggag aaaggatcta tggataagaa atactcaata ggcttagcta tcggcacaaa
      781 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
      841 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
      901 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
      961 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
     1021 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
     1081 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
     1141 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat
     1201 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
     1261 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
     1321 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
     1381 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
     1441 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
     1501 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
     1561 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
     1621 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
     1681 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
     1741 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
     1801 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
     1861 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
     1921 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat
     1981 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
     2041 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
     2101 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
     2161 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
     2221 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
     2281 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
     2341 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
     2401 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
     2461 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
     2521 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
     2581 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
     2641 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
     2701 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca
     2761 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
     2821 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
     2881 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
     2941 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
     3001 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
     3061 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
     3121 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
     3181 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
     3241 tgattatgat gtcgatgcca ttgttccaca aagtttcctt aaagacgatt caatagacaa
     3301 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
     3361 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
     3421 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
     3481 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat
     3541 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
     3601 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
     3661 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
     3721 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
     3781 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
     3841 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
     3901 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
     3961 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
     4021 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
     4081 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
     4141 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
     4201 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
     4261 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
     4321 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
     4381 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
     4441 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
     4501 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
     4561 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
     4621 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
     4681 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
     4741 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
     4801 tctttatgaa acacgcattg atttgagtca gctaggaggt gactgcgcgg gtggcggagg
     4861 aagtgggggc ggtggtagca tgaataaaaa cattgataca gttcgtgaaa ttattactgt
     4921 tgcatctatt ttgattaaat tttccagaga agatattgtt gagaatcgtg ctaattttat
     4981 tgcatttcta aatgagattg gagtaacgca tgaaggtaga aagttaaatc gcaattcatt
     5041 ccgtaaaatt atttctaaat taactcaaga agataagaaa accctcatcg acgaattcaa
     5101 cgagggtttt gagggtgtat atcgatatct agagatgtat acgaacaaat aactcgagta
     5161 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     5221 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     5281 cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac tcgctacgct
     5341 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
     5401 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
     5461 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     5521 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
     5581 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     5641 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     5701 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     5761 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     5821 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     5881 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     5941 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     6001 gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat
     6061 gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg attctcacca
     6121 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
     6181 attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg
     6241 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     6301 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     6361 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     6421 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     6481 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     6541 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     6601 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     6661 catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     6721 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     6781 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     6841 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     6901 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     6961 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccagatat c
//
