LOCUS       sequence-308631-        8073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dxCas9(3.7), MCP-SoxS(R93A/S101A), J306 scRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  AUTHORS   Fontana J, Dong C, Kiattisewee C, Chavali VP, Tickman BI, Carothers 
            JM, Zalatan JG
  TITLE     Effective CRISPRa-mediated control of gene expression in bacteria 
            must overcome strict target site requirements.
  JOURNAL   Nat Commun. 2020 Apr 1;11(1):1618. doi: 10.1038/s41467-020-15454-y.
  PUBMED    32238808
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: "Nat 
            Commun."; date: "2020-04-1"; volume: "11(1):1618. doi"; pages: " 
            10.1038/s41467-020-15454-y"
COMMENT     Sequence Label: pCD565
FEATURES             Location/Qualifiers
     source          1..8073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3925..3996
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4012..4039
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4092..4126
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4136..4147
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      complement(5049..5594)
                     /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(5129..5148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5702..5736
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        5820..5838
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             5861..5890
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5906..5923
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5979..5996)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6149..6195
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(6413..6507)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6528..7187)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7102..7121
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7188..7290)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(7494..7572)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(7868..8073,1..3898)
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gcgcagatat acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga
       61 gatggctaag gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga
      121 ggataaaaag cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca
      181 tgaaaagtac ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc
      241 tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct
      301 catcgagggg gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt
      361 tcagacttac aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa
      421 agcaatcctg agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct
      481 ccctggggag aagaagaacg gcctgtttgg taatcttatc gccctgtccc tcgggctgac
      541 ccccaacttt aaatctaact tcgacctggc cgaagatacc aagcttcaac tgagcaaaga
      601 cacctacgat gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct
      661 ttttttggcg gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa
      721 cacggagatc accaaagctc cgctgagcgc tagtatgatc aagctctatg atgagcacca
      781 ccaagacttg actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga
      841 aattttcttc gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca
      901 ggaggaattt tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct
      961 gctggtaaag cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaat
     1021 catcccccac cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt
     1081 ctaccccttt ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc
     1141 ctactatgta ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc
     1201 agaagagacc atcactccct ggaacttcga gaaagtcgtg gataaggggg cctctgccca
     1261 gtccttcatc gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc
     1321 taaacactct ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata
     1381 cgtcacagaa gggatgagaa agccagcatt cctgtctgga gatcagaaga aagctattgt
     1441 ggacctcctc ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt
     1501 caaaaagatt gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc
     1561 atccctggga acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa
     1621 tgaggagaac gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag
     1681 ggagatgatt gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa
     1741 gcagctcaag aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg
     1801 gatccgagac aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc
     1861 caaccggaac ttcattcagt tgatccatga tgactctctc acctttaagg aggacatcca
     1921 gaaagcacaa gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg
     1981 tagcccagct atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa
     2041 agtaatggga aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac
     2101 cacccagaag ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa
     2161 agaactgggg tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga
     2221 gaagctctac ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga
     2281 catcaatcgg ctctccgact acgacgtgga cgctatcgtg ccccagtctt ttctcaaaga
     2341 tgattctatt gataataaag tgttgacaag atccgataaa aacagaggga agagtgataa
     2401 cgtcccctca gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc
     2461 caaactgatc acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc
     2521 tgagttggat aaagccggtt tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa
     2581 gcacgtggcc caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact
     2641 gattcgagag gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga
     2701 ctttcagttt tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct
     2761 gaatgcagtg gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt
     2821 ttacggagac tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat
     2881 aggcaaggcc accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga
     2941 gattacactg gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac
     3001 aggagaaatc gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat
     3061 gccgcaggtg aacatcgtta aaaagaccga agtacagacc ggaggtttct ccaaggaaag
     3121 tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa
     3181 gaaatacggc ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt
     3241 ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat
     3301 ggagcgatca agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga
     3361 ggtcaaaaaa gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg
     3421 ccggaaacga atgctcgcta gtgcgggcgt gctgcagaaa ggtaacgagc tggcactgcc
     3481 ctctaaatac gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc
     3541 cgaagataat gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat
     3601 catcgagcaa ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa
     3661 ggtgctttct gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat
     3721 tatccacttg tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac
     3781 taccatagac agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca
     3841 tcagtcaatt acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacta
     3901 actcgagtaa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     3961 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     4021 tcgggtgggc ctttctgcgt ttatacctag agcgcccgga agagagtcaa ttcagggtgg
     4081 tgaatctgca gtttacggct agctcagccc taggtattat gctagcgaat tcattaaaga
     4141 ggagaaaggt accatggggc ccgcttctaa ctttactcag ttcgttctcg tcgacaatgg
     4201 cggaactggc gacgtgactg tcgccccaag caacttcgct aacgggatcg ctgaatggat
     4261 cagctctaac tcgcgttcac aggcttacaa agtaacctgt agcgttcgtc agagctctgc
     4321 gcagaatcgc aaatacacca tcaaagtcga ggtgcctaaa ggcgcctggc gttcgtactt
     4381 aaatatggaa ctaaccattc caattttcgc cacgaattcc gactgcgagc ttattgttaa
     4441 ggcaatgcaa ggtctcctaa aagatggaaa cccgattccc tcagcaatcg cagcaaactc
     4501 cggcatctac ggtggcggag gtagcatgtc ccatcagaaa attattcagg atcttatcgc
     4561 atggattgac gagcatattg accagccgct taacattgat gtagtcgcaa aaaaatcagg
     4621 ctattcaaag tggtacttgc aacgaatgtt ccgcacggtg acgcatcaga cgcttggcga
     4681 ttacattcgc caacgccgcc tgttactggc cgccgttgag ttgcgcacca ccgagcgtcc
     4741 gatttttgat atcgcaatgg acctgggtta tgtctcgcag cagaccttct cccgcgtttt
     4801 cgcgcggcag tttgatcgca ctcccgcgga ttatcgccac cgcctgtaag cggccgccac
     4861 gcaaaaaacc ccgcttcggc ggggtttttt cgccctaggg atatattccg cttcctcgct
     4921 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg
     4981 cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca
     5041 aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat
     5101 cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc
     5161 ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg
     5221 cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga
     5281 ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt
     5401 tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg
     5461 gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct
     5521 tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac
     5581 caaaacgatc tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc
     5641 aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag ttgttactag
     5701 attgacagct agctcagtcc taggtataat actagtttgt gtccagaacg ctccgtgttt
     5761 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     5821 catgaggatc acccatgtgc tttttttgaa gcttgggccc gaacaaaaac tcatctcaga
     5881 agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt aaacggtctc
     5941 cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     6001 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     6061 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     6121 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     6181 ggcctttcgt tttatctgtt gtttgtcggt gaactggatc cttactcgag tctagactgc
     6241 aggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6301 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6361 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa actagtgctt ggattctcac
     6421 caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg
     6481 tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta cgccccgccc
     6541 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
     6601 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
     6661 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     6721 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     6781 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     6841 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     6901 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     6961 gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     7021 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     7081 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     7141 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     7201 gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
     7261 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat atcgacgtct
     7321 taagttacga aatcatcctg tggagcttag taggtttagc aagatggcag cgcctaaatg
     7381 tagaatgata aaaggattaa gagattaatt tccctaaaaa tgataaaaca agcgttttga
     7441 aagcgcttgt ttttttggtt tgcagtcaga gtagaataga agtatcaaaa aaagcaccga
     7501 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatgctgtt
     7561 ttgaatggtt ccaacaagat tattttataa cttttataac aaataatcaa ggagaaattc
     7621 aaagaaattt atcagccata aaacaatact taatactata gaatgataac aaaataaact
     7681 actttttaaa agaattttgt gttataatct atttattatt aagtattggg taatattttt
     7741 tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg gaggtcatta
     7801 aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt tattttagga
     7861 ggcaaaaatg gacaagaagt actccattgg gctcgctatc ggcacaaaca gcgtcggctg
     7921 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac
     7981 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac
     8041 ggccgaagcc acgcggctca aaagaacagc acg
//
