LOCUS       sequence-381519-        8073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dxCas9-NG and MCP-SoxS(R93A/S101A) and scRNA J306 on 
            p15A-CmR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  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 8073)
  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: pCK671.306
FEATURES             Location/Qualifiers
     source          1..8073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..548)
                     /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(83..102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        656..690
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        774..792
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             815..844
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             860..877
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(933..950)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1103..1149
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(1367..1461)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1482..2141)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2056..2075
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2142..2244)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(2448..2526)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             2822..6925
                     /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"
     terminator      6952..7023
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7039..7066
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        7119..7153
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             7163..7174
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg
       61 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg
      121 cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg
      181 tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat
      241 gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      301 caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg
      361 agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact
      421 gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa
      481 aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac
      541 gatctcaaga agatcatctt attaatcaga taaaatattt ctagatttca gtgcaattta
      601 tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtta ctagattgac
      661 agctagctca gtcctaggta taatactagt ttgtgtccag aacgctccgt gttttagagc
      721 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcacatgag
      781 gatcacccat gtgctttttt tgaagcttgg gcccgaacaa aaactcatct cagaagagga
      841 tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaacgg tctccagctt
      901 ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga
      961 agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     1021 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg
     1081 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     1141 tcgttttatc tgttgtttgt cggtgaactg gatccttact cgagtctaga ctgcaggctt
     1201 cctcgctcac ttactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1261 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1321 caaaaggcca gcaaaaggcc aggaaccgta aaaaactagt gcttggattc tcaccaataa
     1381 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     1441 ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc gccctgccac
     1501 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     1561 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     1621 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     1681 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     1741 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     1801 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     1861 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     1921 cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     1981 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     2041 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     2101 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     2161 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     2221 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agatatcgac gtcttaagtt
     2281 acgaaatcat cctgtggagc ttagtaggtt tagcaagatg gcagcgccta aatgtagaat
     2341 gataaaagga ttaagagatt aatttcccta aaaatgataa aacaagcgtt ttgaaagcgc
     2401 ttgttttttt ggtttgcagt cagagtagaa tagaagtatc aaaaaaagca ccgactcggt
     2461 gccacttttt caagttgata acggactagc cttattttaa cttgctatgc tgttttgaat
     2521 ggttccaaca agattatttt ataactttta taacaaataa tcaaggagaa attcaaagaa
     2581 atttatcagc cataaaacaa tacttaatac tatagaatga taacaaaata aactactttt
     2641 taaaagaatt ttgtgttata atctatttat tattaagtat tgggtaatat tttttgaaga
     2701 gatattttga aaaagaaaaa ttaaagcata ttaaactaat ttcggaggtc attaaaacta
     2761 ttattgaaat catcaaactc attatggatt taatttaaac tttttatttt aggaggcaaa
     2821 aatggacaag aagtactcca ttgggctcgc tatcggcaca aacagcgtcg gctgggccgt
     2881 cattacggac gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg
     2941 ccacagcata aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga
     3001 agccacgcgg ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg
     3061 ctacctgcag gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag
     3121 gctggaggag tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg
     3181 caatatcgtg gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa
     3241 gaagcttgta gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca
     3301 tatgatcaaa tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga
     3361 tgtcgacaaa ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc
     3421 gatcaacgca tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg
     3481 gcggctcgaa aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa
     3541 tcttatcgcc ctgtccctcg ggctgacccc caactttaaa tctaacttcg acctggccga
     3601 agataccaag cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc
     3661 ccagatcggc gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat
     3721 tctgctgagt gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag
     3781 tatgatcaag ctctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag
     3841 acagcaactg cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc
     3901 cggatacatt gacggcggag caagccagga ggaattttac aaatttatta agcccatctt
     3961 ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg
     4021 caaacagcgc actttcgaca atggaatcat cccccaccag attcacctgg gcgaactgca
     4081 cgctatcctc aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat
     4141 tgagaaaatc ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc
     4201 cagattcgcg tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagaa
     4261 agtcgtggat aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa
     4321 aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt
     4381 ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct
     4441 gtctggagat cagaagaaag ctattgtgga cctcctcttc aagacgaacc ggaaagttac
     4501 cgtgaaacag ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat
     4561 cagcggagtg gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat
     4621 cattaaagac aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt
     4681 cctcaccctt acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc
     4741 tcatctcttc gacgacaaag tcatgaagca gctcaagagg cgccgatata caggatgggg
     4801 gcggctgtca agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct
     4861 ggattttctt aagtccgatg gatttgccaa ccggaacttc attcagttga tccatgatga
     4921 ctctctcacc tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct
     4981 tcacgagcac atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac
     5041 cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt
     5101 tatcgagatg gcccgagaga accaaaccac ccagaaggga cagaagaaca gtagggaaag
     5161 gatgaagagg attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc
     5221 agttgaaaac acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag
     5281 ggacatgtac gtggatcagg aactggacat caatcggctc tccgactacg acgtggacgc
     5341 tatcgtgccc cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc
     5401 cgataaaaac agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa
     5461 aaattattgg cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct
     5521 gactaaggct gaacgaggtg gcctgtctga gttggataaa gccggtttca tcaaaaggca
     5581 gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa
     5641 caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc
     5701 taagctggtc tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa
     5761 ttaccaccat gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa
     5821 atatcccaag cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa
     5881 aatgatcgca aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag
     5941 caatattatg aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg
     6001 accacttatc gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt
     6061 cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt
     6121 acagaccgga ggtttctcca aggaaagtat ccgtccgaaa aggaacagcg acaagctgat
     6181 cgcacgcaaa aaagattggg accccaagaa atacggcgga ttcgtgtctc ctacagtcgc
     6241 ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt
     6301 caaggaactg ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga
     6361 ctttctggag gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa
     6421 gtactctctc tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgcgttttct
     6481 gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag
     6541 ccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga
     6601 acaacacaaa cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt
     6661 gatcctcgcc gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa
     6721 gcccatcagg gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc
     6781 gcctcgtgcc ttcaagtact tcgacactac catagacaga aaggtgtacc gctctacaaa
     6841 ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat
     6901 cgacctctct cagctcggtg gagactaact cgagtaagga tctccaggca tcaaataaaa
     6961 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     7021 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacctagagc
     7081 gcccggaaga gagtcaattc agggtggtga atctgcagtt tacggctagc tcagccctag
     7141 gtattatgct agcgaattca ttaaagagga gaaaggtacc atggggcccg cttctaactt
     7201 tactcagttc gttctcgtcg acaatggcgg aactggcgac gtgactgtcg ccccaagcaa
     7261 cttcgctaac gggatcgctg aatggatcag ctctaactcg cgttcacagg cttacaaagt
     7321 aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt
     7381 gcctaaaggc gcctggcgtt cgtacttaaa tatggaacta accattccaa ttttcgccac
     7441 gaattccgac tgcgagctta ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc
     7501 gattccctca gcaatcgcag caaactccgg catctacggt ggcggaggta gcatgtccca
     7561 tcagaaaatt attcaggatc ttatcgcatg gattgacgag catattgacc agccgcttaa
     7621 cattgatgta gtcgcaaaaa aatcaggcta ttcaaagtgg tacttgcaac gaatgttccg
     7681 cacggtgacg catcagacgc ttggcgatta cattcgccaa cgccgcctgt tactggccgc
     7741 cgttgagttg cgcaccaccg agcgtccgat ttttgatatc gcaatggacc tgggttatgt
     7801 ctcgcagcag accttctccc gcgttttcgc gcggcagttt gatcgcactc ccgcggatta
     7861 tcgccaccgc ctgtaagcgg ccgccacgca aaaaaccccg cttcggcggg gttttttcgc
     7921 cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg
     7981 gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact
     8041 taacagggaa gtgagagggc cgcggcaaag ccg
//
