LOCUS       sequence-189832-        7468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSp Cas9 fused to truncated RTA activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7468)
  AUTHORS   Vora S, Cheng J, Xiao R, VanDusen NJ, Quintino L, Pu WT, 
            Vandenberghe LH, Chavez A, Church G
  TITLE     Rational design of a compact CRISPR-Cas9 activator for AAV-mediated 
            delivery
  JOURNAL   bioRxiv 2018. 298620
REFERENCE   2  (bases 1 to 7468)
  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: "bioRxiv 
            2018. 298620"
COMMENT     Sequence Label: dSp-Rta 125-175
FEATURES             Location/Qualifiers
     source          1..7468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        126..505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        506..709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     660..680
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     706..730
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      818..827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(824..850,851..853,854..3337,3338..3343,3344..3409,
                     3410..3412,3413..4927)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4940..4960
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5198..5217)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5242..5290
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(5604..6192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5684..5703)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6363..7154,7155..7223))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6986..7005
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7224..7328)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7403..7422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
       61 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
      121 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      181 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      241 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      301 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      361 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      421 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      481 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      541 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      601 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      661 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa
      721 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
      781 ccgatccagc ctccggactc tagaggatcg aacccttgcc accatggaca agaagtactc
      841 cattgggctc gctatcggca caaacagcgt cggctgggcc gtcattacgg acgagtacaa
      901 ggtgccgagc aaaaaattca aagttctggg caataccgat cgccacagca taaagaagaa
      961 cctcattggc gccctcctgt tcgactccgg ggagacggcc gaagccacgc ggctcaaaag
     1021 aacagcacgg cgcagatata cccgcagaaa gaatcggatc tgctacctgc aggagatctt
     1081 tagtaatgag atggctaagg tggatgactc tttcttccat aggctggagg agtccttttt
     1141 ggtggaggag gataaaaagc acgagcgcca cccaatcttt ggcaatatcg tggacgaggt
     1201 ggcgtaccat gaaaagtacc caaccatata tcatctgagg aagaagcttg tagacagtac
     1261 tgataaggct gacttgcggt tgatctatct cgcgctggcg catatgatca aatttcgggg
     1321 acacttcctc atcgaggggg acctgaaccc agacaacagc gatgtcgaca aactctttat
     1381 ccaactggtt cagacttaca atcagctttt cgaagagaac ccgatcaacg catccggagt
     1441 tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc cggcggctcg aaaacctcat
     1501 cgcacagctc cctggggaga agaagaacgg cctgtttggt aatcttatcg ccctgtcact
     1561 cgggctgacc cccaacttta aatctaactt cgacctggcc gaagatgcca agcttcaact
     1621 gagcaaagac acctacgatg atgatctcga caatctgctg gcccagatcg gcgaccagta
     1681 cgcagacctt tttttggcgg caaagaacct gtcagacgcc attctgctga gtgatattct
     1741 gcgagtgaac acggagatca ccaaagctcc gctgagcgct agtatgatca agcgctatga
     1801 tgagcaccac caagacttga ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa
     1861 gtacaaggaa attttcttcg atcagtctaa aaatggctac gccggataca ttgacggcgg
     1921 agcaagccag gaggaatttt acaaatttat taagcccatc ttggaaaaaa tggacggcac
     1981 cgaggagctg ctggtaaagc ttaacagaga agatctgttg cgcaaacagc gcactttcga
     2041 caatggaagc atcccccacc agattcacct gggcgaactg cacgctatcc tcaggcggca
     2101 agaggatttc tacccctttt tgaaagataa cagggaaaag attgagaaaa tcctcacatt
     2161 tcggataccc tactatgtag gccccctcgc ccggggaaat tccagattcg cgtggatgac
     2221 tcgcaaatca gaagagacca tcactccctg gaacttcgag gaagtcgtgg ataagggggc
     2281 ctctgcccag tccttcatcg aaaggatgac taactttgat aaaaatctgc ctaacgaaaa
     2341 ggtgcttcct aaacactctc tgctgtacga gtacttcaca gtttataacg agctcaccaa
     2401 ggtcaaatac gtcacagaag ggatgagaaa gccagcattc ctgtctggag agcagaagaa
     2461 agctatcgtg gacctcctct tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga
     2521 agactatttc aaaaagattg aatgtttcga ctctgttgaa atcagcggag tggaggatcg
     2581 cttcaacgca tccctgggaa cgtatcacga tctcctgaaa atcattaaag acaaggactt
     2641 cctggacaat gaggagaacg aggacattct tgaggacatt gtcctcaccc ttacgttgtt
     2701 tgaagatagg gagatgattg aagaacgctt gaaaacttac gctcatctct tcgacgacaa
     2761 agtcatgaaa cagctcaaga ggcgccgata tacaggatgg gggcggctgt caagaaaact
     2821 gatcaatggg atccgagaca agcagagtgg aaagacaatc ctggattttc ttaagtccga
     2881 tggatttgcc aaccggaact tcatgcagtt gatccatgat gactctctca cctttaagga
     2941 ggacatccag aaagcacaag tttctggcca gggggacagt cttcacgagc acatcgctaa
     3001 tcttgcaggt agcccagcta tcaaaaaggg aatactgcag accgttaagg tcgtggatga
     3061 actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc gttatcgaga tggcccgaga
     3121 gaaccaaact acccagaagg gacagaagaa cagtagggaa aggatgaaga ggattgaaga
     3181 gggtataaaa gaactggggt cccaaatcct taaggaacac ccagttgaaa acacccagct
     3241 tcagaatgag aagctctacc tgtactacct gcagaacggc agggacatgt acgtggatca
     3301 ggaactggac atcaatcggc tctccgacta cgacgtggct gctatcgtgc cccagtcttt
     3361 tctcaaagat gattctattg ataataaagt gttgacaaga tccgataaag ctagagggaa
     3421 gagtgataac gtcccctcag aagaagttgt caagaaaatg aaaaattatt ggcggcagct
     3481 gctgaacgcc aaactgatca cacaacggaa gttcgataat ctgactaagg ctgaacgagg
     3541 tggcctgtct gagttggata aagccggctt catcaaaagg cagcttgttg agacacgcca
     3601 gatcaccaag cacgtggccc aaattctcga ttcacgcatg aacaccaagt acgatgaaaa
     3661 tgacaaactg attcgagagg tgaaagttat tactctgaag tctaagctgg tctcagattt
     3721 cagaaaggac tttcagtttt ataaggtgag agagatcaac aattaccacc atgcgcatga
     3781 tgcctacctg aatgcagtgg taggcactgc acttatcaaa aaatatccca agcttgaatc
     3841 tgaatttgtt tacggagact ataaagtgta cgatgttagg aaaatgatcg caaagtctga
     3901 gcaggaaata ggcaaggcca ccgctaagta cttcttttac agcaatatta tgaatttttt
     3961 caagaccgag attacactgg ccaatggaga gattcggaag cgaccactta tcgaaacaaa
     4021 cggagaaaca ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag tccggaaggt
     4081 cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg gaggcttctc
     4141 caaggaaagt atcctcccga aaaggaacag cgacaagctg atcgcacgca aaaaagattg
     4201 ggaccccaag aaatacggcg gattcgattc tcctacagtc gcttacagtg tactggttgt
     4261 ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat
     4321 cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg
     4381 atataaagag gtcaaaaaag acctcatcat taagcttccc aagtactctc tctttgagct
     4441 tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct
     4501 ggcactgccc tctaaatacg ttaatttctt gtatctggcc agccactatg aaaagctcaa
     4561 agggtctccc gaagataatg agcagaagca gctgttcgtg gaacaacaca aacactacct
     4621 tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa
     4681 cctcgataag gtgctttctg cttacaataa gcacagggat aagcccatca gggagcaggc
     4741 agaaaacatt atccacttgt ttactctgac caacttgggc gcgcctgcag ccttcaagta
     4801 cttcgacacc accatagaca gaaagcggta cacctctaca aaggaggtcc tggacgccac
     4861 actgattcat cagtcaatta cggggctcta tgaaacaaga atcgacctct ctcagctcgg
     4921 tggagacagc agggctgacc ccaagaagaa gaggaaggtg agtggtggag gaagtggcgg
     4981 gtcagggtcg gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac
     5041 acttgagtcc atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga
     5101 gattctggat accttcctga acgacgagtg cctcttgcat gcctagaaga attcgatccc
     5161 taccggttag taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac
     5221 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
     5281 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5341 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5401 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctcc atagtcggtc
     5461 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5521 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5581 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5641 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5701 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5761 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5821 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5881 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5941 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6001 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6061 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6121 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6181 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6241 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6301 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6361 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6421 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6481 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6541 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6601 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6661 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6721 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6781 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6841 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6901 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6961 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7021 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7081 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7141 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7201 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7261 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7321 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     7381 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     7441 tgctccctgc ttgtgtgttg gaggtcgc
//
