LOCUS       sequence-192081-        8278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSp Cas9 fused to combination of truncated activation 
            domains.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8278)
  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 8278)
  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-p65(100-261)-RTA(125-190)
FEATURES             Location/Qualifiers
     source          1..8278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(748..774,775..777,778..3261,3262..3267,3268..3333,
                     3334..3336,3337..4851)
                     /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             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4939..5088
                     /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             5113..5133
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5293..5649
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     primer_bind     complement(5932..5951)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5976..6024
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(6338..6926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6418..6437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7097..7888,7889..7957))
                     /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     7720..7739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7958..8062)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8137..8156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc
      781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
      961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1261 ggggacctga acccagacaa cagcgatgtc gataaactct ttatccaact ggttcagact
     1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3241 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct
     3301 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc
     3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4861 gaccccaaga agaagaggaa ggtgagtggt ggaggaagtg gcgggtcagg gtcggaggcc
     4921 agcggttccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac
     4981 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc
     5041 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctaga
     5101 agttccggat ctccgaaaaa gaaacgcaaa gttggttcgg gaggtggttc gggtggctct
     5161 ggatcagtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt gtctgcactg
     5221 gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc tgtggctcca
     5281 ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct gctgcagctg
     5341 cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc
     5401 accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca gggcatccct
     5461 gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat cacccggctc
     5521 gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc accaggcctg
     5581 cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgatat ggatttctca
     5641 gccttgctgt caggcggtgg tagtggtggg agcggtagtg acctttccca tccgccccca
     5701 aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga tctgaacctg
     5761 gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa cgacgagtgc
     5821 ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc tctgttttag
     5881 aagaattcga tccctaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     5941 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     6001 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     6061 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     6121 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     6181 ctccatagtc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6241 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6301 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6361 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6421 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6481 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6541 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6601 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6661 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6721 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6781 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6841 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6901 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6961 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7021 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7081 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7141 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7201 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7261 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7321 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7381 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7441 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7501 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7561 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7621 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7681 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7741 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7801 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7861 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7921 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7981 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     8101 cgggagatct cccgatcccc tatggtcgac tctcagtaca atctgctctg atgccgcata
     8161 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     8221 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagg
//
