LOCUS       sequence-381516-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dxCas9-NG and MCP-SoxS(R93A/S101A) on p15A-CmR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7368)
  AUTHORS   Kiattisewee C, Karanjia AV, Legut M, Daniloski Z, Koplik SE, Nelson 
            J, Kleinstiver BP, Sanjana NE, Carothers JM, Zalatan JG
  TITLE     Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible
            dCas9 Variants.
  JOURNAL   ACS Synth Biol. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405.
  PUBMED    36378874
REFERENCE   2  (bases 1 to 7368)
  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. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405."
COMMENT     Sequence Label: pCK669
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2826..2897
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2913..2940
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2993..3027
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             3037..3048
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3816..3833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3950..4495)
                     /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(4030..4049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4609..4703)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4724..5383)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5298..5317
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5384..5486)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(5690..5768)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(6064..7368,1..2799)
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc
       61 gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc
      121 tggaacttcg agaaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg
      181 actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac
      241 gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga
      301 aagccagcat tcctgtctgg agatcagaag aaagctattg tggacctcct cttcaagacg
      361 aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc
      421 gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac
      481 gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt
      541 cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc
      601 ttgaaaactt acgctcatct cttcgacgac aaagtcatga agcagctcaa gaggcgccga
      661 tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga caagcagagt
      721 ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcattcag
      781 ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc
      841 cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag
      901 ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag
      961 cccgagaata tcgttatcga gatggcccga gagaaccaaa ccacccagaa gggacagaag
     1021 aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc
     1081 cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac
     1141 ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac
     1201 tacgacgtgg acgctatcgt gccccagtct tttctcaaag atgattctat tgataataaa
     1261 gtgttgacaa gatccgataa aaacagaggg aagagtgata acgtcccctc agaagaagtt
     1321 gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg
     1381 aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggt
     1441 ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc
     1501 gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt
     1561 attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg
     1621 agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact
     1681 gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg
     1741 tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag
     1801 tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga
     1861 gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac
     1921 aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt
     1981 aaaaagaccg aagtacagac cggaggtttc tccaaggaaa gtatccgtcc gaaaaggaac
     2041 agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgtg
     2101 tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa
     2161 aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa
     2221 aaaaacccca tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc
     2281 attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct
     2341 agtgcgcgtt ttctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc
     2401 ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag
     2461 cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgaa
     2521 ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat
     2581 aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg
     2641 accaacttgg gcgcgcctcg tgccttcaag tacttcgaca ctaccataga cagaaaggtg
     2701 taccgctcta caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc
     2761 tatgaaacaa gaatcgacct ctctcagctc ggtggagact aactcgagta aggatctcca
     2821 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     2881 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     2941 tttataccta gagcgcccgg aagagagtca attcagggtg gtgaatctgc agtttacggc
     3001 tagctcagcc ctaggtatta tgctagcgaa ttcattaaag aggagaaagg taccatgggg
     3061 cccgcttcta actttactca gttcgttctc gtcgacaatg gcggaactgg cgacgtgact
     3121 gtcgccccaa gcaacttcgc taacgggatc gctgaatgga tcagctctaa ctcgcgttca
     3181 caggcttaca aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc
     3241 atcaaagtcg aggtgcctaa aggcgcctgg cgttcgtact taaatatgga actaaccatt
     3301 ccaattttcg ccacgaattc cgactgcgag cttattgtta aggcaatgca aggtctccta
     3361 aaagatggaa acccgattcc ctcagcaatc gcagcaaact ccggcatcta cggtggcgga
     3421 ggtagcatgt cccatcagaa aattattcag gatcttatcg catggattga cgagcatatt
     3481 gaccagccgc ttaacattga tgtagtcgca aaaaaatcag gctattcaaa gtggtacttg
     3541 caacgaatgt tccgcacggt gacgcatcag acgcttggcg attacattcg ccaacgccgc
     3601 ctgttactgg ccgccgttga gttgcgcacc accgagcgtc cgatttttga tatcgcaatg
     3661 gacctgggtt atgtctcgca gcagaccttc tcccgcgttt tcgcgcggca gtttgatcgc
     3721 actcccgcgg attatcgcca ccgcctgtaa gcggccgcca cgcaaaaaac cccgcttcgg
     3781 cggggttttt tcgccctagg gatatattcc gcttcctcgc tcactgactc gctacgctcg
     3841 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat
     3901 gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc
     3961 tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     4021 caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc
     4081 ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc
     4141 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
     4201 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
     4261 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
     4321 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
     4381 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa
     4441 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
     4501 tcatcttatt aatcagataa aatatttcta gatttcagtg caatttatct cttcaaatgt
     4561 agcacctgaa gtcagcccca tacgatataa gttgttacta gtgcttggat tctcaccaat
     4621 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     4681 tactggatct atcaacagga gtccaagcga gctcgatatc aaattacgcc ccgccctgcc
     4741 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     4801 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     4861 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     4921 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     4981 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact
     5041 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     5101 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     5161 tacgaaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     5221 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     5281 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     5341 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc
     5401 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     5461 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccagatatcg acgtcttaag
     5521 ttacgaaatc atcctgtgga gcttagtagg tttagcaaga tggcagcgcc taaatgtaga
     5581 atgataaaag gattaagaga ttaatttccc taaaaatgat aaaacaagcg ttttgaaagc
     5641 gcttgttttt ttggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg
     5701 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga
     5761 atggttccaa caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag
     5821 aaatttatca gccataaaac aatacttaat actatagaat gataacaaaa taaactactt
     5881 tttaaaagaa ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa
     5941 gagatatttt gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac
     6001 tattattgaa atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca
     6061 aaaatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc
     6121 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
     6181 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc
     6241 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
     6301 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     6361 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     6421 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     6481 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     6541 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     6601 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     6661 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     6721 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     6781 aatcttatcg ccctgtccct cgggctgacc cccaacttta aatctaactt cgacctggcc
     6841 gaagatacca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     6901 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc
     6961 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     7021 agtatgatca agctctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     7081 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     7141 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     7201 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     7261 cgcaaacagc gcactttcga caatggaatc atcccccacc agattcacct gggcgaactg
     7321 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagat
//
