LOCUS       sequence-192088-        8137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSp Cas9 fused at C term. To VP64 and RTA activation 
            domains.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8137)
  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 8137)
  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-VP64-RTA
FEATURES             Location/Qualifiers
     source          1..8137
                     /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"
     CDS             4999..5148
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5173..5193
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5227..5796
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     primer_bind     complement(5851..5870)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5895..5943
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(6257..6845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6337..6356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7016..7807,7808..7876))
                     /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     7639..7658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7877..7981)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8056..8075)
                     /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 gtcggaggcc
     4981 agcggttccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac
     5041 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc
     5101 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctaga
     5161 agttccggat ctccgaaaaa gaaacgcaaa gttggttcgg gaggtggttc gggtggctct
     5221 ggatcacggg attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt
     5281 agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct
     5341 ccaggaagtc catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt
     5401 ccagtacatg agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca
     5461 gcgcccgcag tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc
     5521 caggctgtca aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct
     5581 gcaatctgtg gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg
     5641 acaaccacac ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa
     5701 ttgaacgaga ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc
     5761 agcacaggac tgtccatctt cgacacatct ctgttttaga agaattcgat ccctaccggt
     5821 tagtaatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     5881 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg
     5941 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     6001 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     6061 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tccatagtcg gtcgttcggc
     6121 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6181 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6241 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6301 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6361 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6421 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6481 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6541 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6601 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6661 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6721 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6781 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6841 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6901 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6961 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7021 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7081 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7141 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7201 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7261 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7321 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7381 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7441 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7501 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7561 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7621 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7681 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7741 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7801 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7861 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7921 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7981 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     8041 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     8101 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagc
//
