LOCUS       sequence-192044-        7540 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 7540)
  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 7540)
  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 100-175
FEATURES             Location/Qualifiers
     source          1..7540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     690..714
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      802..811
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(808..834,835..837,838..3321,3322..3327,3328..3393,
                     3394..3396,3397..4911)
                     /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             4924..4944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5254..5273)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5298..5346
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(5660..6248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5740..5759)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6419..7210,7211..7279))
                     /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     7042..7061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7280..7384)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7459..7478)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      721 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      781 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc
      841 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      901 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      961 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
     1021 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1081 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1141 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1201 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1261 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1321 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1381 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1441 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1501 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1561 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1621 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1681 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1741 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1801 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1861 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1921 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1981 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     2041 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2101 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2161 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2221 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2281 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2341 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2401 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2461 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2521 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2581 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2641 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2701 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2761 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2821 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2881 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2941 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     3001 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3061 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3121 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3181 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3241 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3301 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct
     3361 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc
     3421 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3481 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3541 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3601 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3661 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3721 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3781 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3841 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3901 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3961 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     4021 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4081 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4141 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4201 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4261 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4321 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4381 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4441 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4501 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4561 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4621 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4681 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4741 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4801 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4861 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4921 gaccccaaga agaagaggaa ggtgagtggt ggaggaagtg gcgggtcagg gtcggtcaaa
     4981 gcccttcggg agatggccga tactgtgatt ccccagaagg aagaggctgc aatctgtggc
     5041 caaatggacc tttcccatcc gcccccaagg ggccatctgg atgagctgac aaccacactt
     5101 gagtccatga ccgaggatct gaacctggac tcacccctga ccccggaatt gaacgagatt
     5161 ctggatacct tcctgaacga cgagtgcctc ttgcatgcct agaagaattc gatccctacc
     5221 ggttagtaat gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga
     5281 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt
     5341 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5401 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5461 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctccatag tcggtcgttc
     5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6061 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7381 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     7441 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7501 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
//
