LOCUS       sequence-486128-        7859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of dCas9-AsiA and constitutive expression 
            of MCP-SoxS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7859)
  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 7859)
  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: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK321
FEATURES             Location/Qualifiers
     source          1..7859
                     /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"
     CDS             complement(1020..1646)
                     /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     1551..1570
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1662..1717
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1698..1716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1735..1746
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1753..1779,1780..1782,1783..4269,4270..4272,
                     4273..5856)
                     /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      6189..6260
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6276..6303
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        6356..6390
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             6400..6411
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(7179..7196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7313..7858)
                     /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(7393..7412)
                     /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 taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa ttcaaggccg
     1081 aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg tcgtaataat
     1141 ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg atgctcttga
     1201 tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata taatgcattc
     1261 tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt ttcatactgt
     1321 ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact tagtaaagca
     1381 catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc ttctaaaggg
     1441 caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag caaagcccgc
     1501 ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg ggcgagttta
     1561 cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct gttaatcact
     1621 ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact ctatcgttga
     1681 tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag atctaaagag
     1741 gagaaaggat ctatggataa gaaatactca ataggcttag ctatcggcac aaatagcgtc
     1801 ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga
     1861 aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga
     1921 gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac acgtcggaag
     1981 aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt
     2041 ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat
     2101 cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat
     2161 catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg
     2221 gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct
     2281 gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt
     2341 gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg
     2401 agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc
     2461 ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt
     2521 gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat
     2581 aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta
     2641 tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc
     2701 ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa
     2761 gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa
     2821 aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc
     2881 aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa
     2941 gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg
     3001 ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat
     3061 cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg
     3121 cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg
     3181 aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca
     3241 aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag
     3301 tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa
     3361 ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat
     3421 cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat
     3481 agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat
     3541 ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta
     3601 gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt
     3661 aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat
     3721 actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc
     3781 aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg
     3841 atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa
     3901 ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt
     3961 attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca
     4021 gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat
     4081 tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt
     4141 aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc
     4201 caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat
     4261 gatgtcgatg ccattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc
     4321 ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc
     4381 aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag
     4441 tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt
     4501 atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat
     4561 agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt
     4621 accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt
     4681 gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct
     4741 ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat
     4801 gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat
     4861 ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag
     4921 attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa
     4981 gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag
     5041 aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg
     5101 gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt
     5161 ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag
     5221 ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa
     5281 aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt
     5341 aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt
     5401 gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta
     5461 tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa
     5521 ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt
     5581 tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa
     5641 catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg
     5701 aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat
     5761 acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat
     5821 gaaacacgca ttgatttgag tcagctagga ggtgactgcg cgggtggcgg aggaagtggg
     5881 ggcggtggta gcatgaataa aaacattgat acagttcgtg aaattattac tgttgcatct
     5941 attttgatta aattttccag agaagatatt gttgagaatc gtgctaattt tattgcattt
     6001 ctaaatgaga ttggagtaac gcatgaaggt agaaagttaa atcgcaattc attccgtaaa
     6061 attatttcta aattaactca agaagataag aaaaccctca tcgacgaatt caacgagggt
     6121 tttgagggtg tatatcgata tctagagatg tatacgaaca aataactcga gtaaggatct
     6181 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     6241 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     6301 gcgtttatac ctagagcgcc cggaagagag tcaattcagg gtggtgaatc tgcagtttac
     6361 ggctagctca gccctaggta ttatgctagc gaattcatta aagaggagaa aggtaccatg
     6421 gggcccgctt ctaactttac tcagttcgtt ctcgtcgaca atggcggaac tggcgacgtg
     6481 actgtcgccc caagcaactt cgctaacggg atcgctgaat ggatcagctc taactcgcgt
     6541 tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct ctgcgcagaa tcgcaaatac
     6601 accatcaaag tcgaggtgcc taaaggcgcc tggcgttcgt acttaaatat ggaactaacc
     6661 attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc
     6721 ctaaaagatg gaaacccgat tccctcagca atcgcagcaa actccggcat ctacggtggc
     6781 ggaggtagca tgtcccatca gaaaattatt caggatctta tcgcatggat tgacgagcat
     6841 attgaccagc cgcttaacat tgatgtagtc gcaaaaaaat caggctattc aaagtggtac
     6901 ttgcaacgaa tgttccgcac ggtgacgcat cagacgcttg gcgattacat tcgccaacgc
     6961 cgcctgttac tggccgccgt tgagttgcgc accaccgagc gtccgatttt tgatatcgca
     7021 atggacctgg gttatgtctc gcagcagacc ttctcccgcg ttttcgcgcg gcagtttgat
     7081 cgcactcccg cggattatcg ccaccgcctg taagcggccg ccacgcaaaa aaccccgctt
     7141 cggcggggtt ttttcgccct agggatatat tccgcttcct cgctcactga ctcgctacgc
     7201 tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa
     7261 gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata
     7321 ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     7381 cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg
     7441 ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca
     7501 cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc
     7561 cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa
     7621 agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt
     7681 gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc
     7741 aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg
     7801 caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaag
//
