LOCUS       sequence-296070-        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"
     misc_RNA        complement(209..287)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             583..4686
                     /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"
     terminator      4713..4784
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4800..4827
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4880..4914
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             4924..4935
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      complement(5837..6382)
                     /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(5917..5936)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6490..6524
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        6608..6626
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             6649..6678
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6694..6711
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6767..6784)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6937..6983
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(7201..7295)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7316..7975)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7890..7909
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(7976..8073,1..5))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gatcaacgtc tcattttcgc cagatatcga cgtcttaagt tacgaaatca tcctgtggag
       61 cttagtaggt ttagcaagat ggcagcgcct aaatgtagaa tgataaaagg attaagagat
      121 taatttccct aaaaatgata aaacaagcgt tttgaaagcg cttgtttttt tggtttgcag
      181 tcagagtaga atagaagtat caaaaaaagc accgactcgg tgccactttt tcaagttgat
      241 aacggactag ccttatttta acttgctatg ctgttttgaa tggttccaac aagattattt
      301 tataactttt ataacaaata atcaaggaga aattcaaaga aatttatcag ccataaaaca
      361 atacttaata ctatagaatg ataacaaaat aaactacttt ttaaaagaat tttgtgttat
      421 aatctattta ttattaagta ttgggtaata ttttttgaag agatattttg aaaaagaaaa
      481 attaaagcat attaaactaa tttcggaggt cattaaaact attattgaaa tcatcaaact
      541 cattatggat ttaatttaaa ctttttattt taggaggcaa aaatggacaa gaagtactcc
      601 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag
      661 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac
      721 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga
      781 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt
      841 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg
      901 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg
      961 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact
     1021 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga
     1081 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc
     1141 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt
     1201 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc
     1261 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtccctc
     1321 gggctgaccc ccaactttaa atctaacttc gacctggccg aagataccaa gcttcaactg
     1381 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac
     1441 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg
     1501 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gctctatgat
     1561 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag
     1621 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga
     1681 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc
     1741 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac
     1801 aatggaatca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa
     1861 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt
     1921 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact
     1981 cgcaaatcag aagagaccat cactccctgg aacttcgaga aagtcgtgga taagggggcc
     2041 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag
     2101 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag
     2161 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga tcagaagaaa
     2221 gctattgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa
     2281 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc
     2341 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc
     2401 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt
     2461 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa
     2521 gtcatgaagc agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg
     2581 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat
     2641 ggatttgcca accggaactt cattcagttg atccatgatg actctctcac ctttaaggag
     2701 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat
     2761 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa
     2821 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag
     2881 aaccaaacca cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag
     2941 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt
     3001 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag
     3061 gaactggaca tcaatcggct ctccgactac gacgtggacg ctatcgtgcc ccagtctttt
     3121 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa cagagggaag
     3181 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg
     3241 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt
     3301 ggcctgtctg agttggataa agccggtttc atcaaaaggc agcttgttga gacacgccag
     3361 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat
     3421 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc
     3481 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat
     3541 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct
     3601 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag
     3661 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc
     3721 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac
     3781 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc
     3841 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggtttctcc
     3901 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg
     3961 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg
     4021 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc
     4081 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga
     4141 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt
     4201 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgtgc tgcagaaagg taacgagctg
     4261 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa
     4321 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt
     4381 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac
     4441 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca
     4501 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac
     4561 ttcgacacta ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca
     4621 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt
     4681 ggagactaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag
     4741 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     4801 gctcaccttc gggtgggcct ttctgcgttt atacctagag cgcccggaag agagtcaatt
     4861 cagggtggtg aatctgcagt ttacggctag ctcagcccta ggtattatgc tagcgaattc
     4921 attaaagagg agaaaggtac catggggccc gcttctaact ttactcagtt cgttctcgtc
     4981 gacaatggcg gaactggcga cgtgactgtc gccccaagca acttcgctaa cgggatcgct
     5041 gaatggatca gctctaactc gcgttcacag gcttacaaag taacctgtag cgttcgtcag
     5101 agctctgcgc agaatcgcaa atacaccatc aaagtcgagg tgcctaaagg cgcctggcgt
     5161 tcgtacttaa atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt
     5221 attgttaagg caatgcaagg tctcctaaaa gatggaaacc cgattccctc agcaatcgca
     5281 gcaaactccg gcatctacgg tggcggaggt agcatgtccc atcagaaaat tattcaggat
     5341 cttatcgcat ggattgacga gcatattgac cagccgctta acattgatgt agtcgcaaaa
     5401 aaatcaggct attcaaagtg gtacttgcaa cgaatgttcc gcacggtgac gcatcagacg
     5461 cttggcgatt acattcgcca acgccgcctg ttactggccg ccgttgagtt gcgcaccacc
     5521 gagcgtccga tttttgatat cgcaatggac ctgggttatg tctcgcagca gaccttctcc
     5581 cgcgttttcg cgcggcagtt tgatcgcact cccgcggatt atcgccaccg cctgtaagcg
     5641 gccgccacgc aaaaaacccc gcttcggcgg ggttttttcg ccctagggat atattccgct
     5701 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     5761 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     5821 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     5881 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5941 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     6001 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     6061 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     6121 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     6181 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     6241 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     6301 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     6361 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat
     6421 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     6481 gttactagat tgacagctag ctcagtccta ggtataatac tagtttgtgt ccagaacgct
     6541 ccgtgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     6601 aagtggcaca tgaggatcac ccatgtgctt tttttgaagc ttgggcccga acaaaaactc
     6661 atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttaa
     6721 acggtctcca gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     6781 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     6841 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     6901 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     6961 aaagactggg cctttcgttt tatctgttgt ttgtcggtga actggatcct tactcgagtc
     7021 tagactgcag gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     7081 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     7141 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaaac tagtgcttgg
     7201 attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag
     7261 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg
     7321 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     7381 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     7441 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     7501 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
     7561 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     7621 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     7681 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     7741 ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
     7801 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     7861 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     7921 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
     7981 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     8041 cattatggtg aaagttggaa cctcttacgt gcc
//
