LOCUS       sequence-189821-        7615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSp Cas9 fused to truncated p65 activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  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 7615)
  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-p65 (1-100)
FEATURES             Location/Qualifiers
     source          1..7615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        146..525
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        526..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      838..847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(844..870,871..873,874..3357,3358..3363,3364..3429,
                     3430..3432,3433..4947)
                     /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             4960..4980
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5365..5384)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5409..5457
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(5771..6359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5851..5870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6530..7321,7322..7390))
                     /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     7153..7172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7391..7495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7570..7589)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
       61 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
      121 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      181 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      241 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      301 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      361 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      421 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      481 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      541 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      601 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      661 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      781 ctccatagaa gacaccggga ccgatccagc ctccggactc tagaggatcg aacccttgcc
      841 accatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc
      901 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
      961 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc
     1021 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
     1081 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     1141 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     1201 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     1261 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     1321 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     1381 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     1441 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     1501 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     1561 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc
     1621 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     1681 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc
     1741 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     1801 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     1861 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     1921 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     1981 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     2041 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg
     2101 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag
     2161 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat
     2221 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag
     2281 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat
     2341 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca
     2401 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc
     2461 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt
     2521 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa
     2581 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa
     2641 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt
     2701 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac
     2761 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg
     2821 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc
     2881 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat
     2941 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt
     3001 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag
     3061 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc
     3121 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa
     3181 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac
     3241 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc
     3301 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggct
     3361 gctatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga
     3421 tccgataaag ctagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg
     3481 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat
     3541 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg
     3601 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg
     3661 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag
     3721 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac
     3781 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa
     3841 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg
     3901 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac
     3961 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag
     4021 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat
     4081 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa
     4141 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg
     4201 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc
     4261 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc
     4321 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc
     4381 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc
     4441 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag
     4501 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc
     4561 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg
     4621 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga
     4681 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat
     4741 aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc
     4801 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca
     4861 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga
     4921 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg
     4981 agtggtggag gaagtggcgg gtcagggtcg agccagtacc tgcccgacac cgacgaccgg
     5041 caccggatcg aggaaaagcg gaagcggacc tacgagacat tcaagagcat catgaagaag
     5101 tcccccttca gcggccccac cgaccctaga cctccaccta gaagaatcgc cgtgcccagc
     5161 agatccagcg ccagcgtgcc aaaacctgcc ccccagcctt accccttcac cagcagcctg
     5221 agcaccatca actacgacga gttccctacc atggtgttcc ccagcggcca gatctctcag
     5281 gcctctgctc tggctccagc ccctcctcag tagaagaatt cgatccctac cggttagtaa
     5341 tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     5401 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     5461 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     5521 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     5581 cagggctcgc agccaacgtc ggggcggcag gccctccata gtcggtcgtt cggctgcggc
     5641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6181 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6541 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6601 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6661 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6721 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6781 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6841 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6901 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6961 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7021 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7081 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7141 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7201 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7261 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7321 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7381 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7501 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     7561 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccct
//
