LOCUS       sequence-127065-        4116 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  This plasmid contains a Cas9 Activator for yeast. The activator is 
            about 1.2-1.8 X more potent than just dCas9-VP64 fusion in yeast. It
            is on a single copy CEN/ARS plasmid with Ura marker, pRS416..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4116)
  AUTHORS   Naranjo S, Smith JD, Artieri CG, Zhang M, Zhou Y, Palmer ME, Fraser 
            HB
  TITLE     Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation.
  JOURNAL   PLoS Genet. 2015 Dec 29;11(12):e1005751. doi: 
            10.1371/journal.pgen.1005751. eCollection 2015 Dec.
  PUBMED    26713447
REFERENCE   2  (bases 1 to 4116)
  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: "PLoS 
            Genet."; date: "2015-12-29"; volume: "11(12):e1005751. doi"; pages: 
            " 10.1371/journal.pgen.1005751. eCollection 2015 Dec"
COMMENT     Sequence Label: pRS416-Gal4-dCas9-VP64
FEATURES             Location/Qualifiers
     source          1..4116
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(join(13..1527,1528..1530,1531..1596,1597..1602,
                     1603..4086,4087..4089,4090..4116))
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(join(13..1596,1597..1599,1600..4086,4087..4089,
                     4090..4116))
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(13..4116)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gtcagccctg ctgtctccac cgagctgaga gaggtcgatt cttgtttcat agagccccgt
       61 aattgactga tgaatcagtg tggcgtccag gacctccttt gtagaggtgt accgctttct
      121 gtctatggtg gtgtcgaagt acttgaaggc tgcaggcgcg cccaagttgg tcagagtaaa
      181 caagtggata atgttttctg cctgctccct gatgggctta tccctgtgct tattgtaagc
      241 agaaagcacc ttatcgaggt tagcgtcggc gaggatcact cttttggaga attcgcttat
      301 ttgctcgatg atctcatcaa ggtagtgttt gtgttgttcc acgaacagct gcttctgctc
      361 attatcttcg ggagaccctt tgagcttttc atagtggctg gccagataca agaaattaac
      421 gtatttagag ggcagtgcca gctcgttacc tttctgcagc tcgcccgcac tagcgagcat
      481 tcgtttccgg ccgttttcaa gctcaaagag agagtacttg ggaagcttaa tgatgaggtc
      541 ttttttgacc tctttatatc ctttcgcctc gagaaagtcg atggggtttt tttcgaagct
      601 tgatcgctcc atgattgtga tgcccagcag ttccttgacg cttttgagtt ttttagactt
      661 ccctttctcc actttggcca caaccagtac actgtaagcg actgtaggag aatcgaatcc
      721 gccgtatttc ttggggtccc aatctttttt gcgtgcgatc agcttgtcgc tgttcctttt
      781 cgggaggata ctttccttgg agaagcctcc ggtctgtact tcggtctttt taacgatgtt
      841 cacctgcggc atggacagga ccttccggac tgtcgcgaaa tccctaccct tgtcccacac
      901 gatttctcct gtttctccgt ttgtttcgat aagtggtcgc ttccgaatct ctccattggc
      961 cagtgtaatc tcggtcttga aaaaattcat aatattgctg taaaagaagt acttagcggt
     1021 ggccttgcct atttcctgct cagactttgc gatcattttc ctaacatcgt acactttata
     1081 gtctccgtaa acaaattcag attcaagctt gggatatttt ttgataagtg cagtgcctac
     1141 cactgcattc aggtaggcat catgcgcatg gtggtaattg ttgatctctc tcaccttata
     1201 aaactgaaag tcctttctga aatctgagac cagcttagac ttcagagtaa taactttcac
     1261 ctctcgaatc agtttgtcat tttcatcgta cttggtgttc atgcgtgaat cgagaatttg
     1321 ggccacgtgc ttggtgatct ggcgtgtctc aacaagctgc cttttgatga agccggcttt
     1381 atccaactca gacaggccac ctcgttcagc cttagtcaga ttatcgaact tccgttgtgt
     1441 gatcagtttg gcgttcagca gctgccgcca ataatttttc attttcttga caacttcttc
     1501 tgaggggacg ttatcactct tccctctatt tttatcggat cttgtcaaca ctttattatc
     1561 aatagaatca tctttgagaa aagactgggg cacgatagca tccacgtcgt agtcggagag
     1621 ccgattgatg tccagttcct gatccacgta catgtccctg ccgttctgca ggtagtacag
     1681 gtagagcttc tcattctgaa gctgggtgtt ttcaactggg tgttccttaa ggatttggga
     1741 ccccagttct tttataccct cttcaatcct cttcatcctt tccctactgt tcttctgtcc
     1801 cttctgggta gtttggttct ctcgggccat ctcgataacg atattctcgg gcttatgcct
     1861 tcccattact ttgacgagtt catccacgac cttaacggtc tgcagtattc cctttttgat
     1921 agctgggcta cctgcaagat tagcgatgtg ctcgtgaaga ctgtccccct ggccagaaac
     1981 ttgtgctttc tggatgtcct ccttaaaggt gagagagtca tcatggatca actgcatgaa
     2041 gttccggttg gcaaatccat cggacttaag aaaatccagg attgtctttc cactctgctt
     2101 gtctcggatc ccattgatca gttttcttga cagccgcccc catcctgtat atcggcgcct
     2161 cttgagctgt ttcatgactt tgtcgtcgaa gagatgagcg taagttttca agcgttcttc
     2221 aatcatctcc ctatcttcaa acaacgtaag ggtgaggaca atgtcctcaa gaatgtcctc
     2281 gttctcctca ttgtccagga agtccttgtc tttaatgatt ttcaggagat cgtgatacgt
     2341 tcccagggat gcgttgaagc gatcctccac tccgctgatt tcaacagagt cgaaacattc
     2401 aatctttttg aaatagtctt ctttgagctg tttcacggta actttccggt tcgtcttgaa
     2461 gaggaggtcc acgatagctt tcttctgctc tccagacagg aatgctggct ttctcatccc
     2521 ttctgtgacg tatttgacct tggtgagctc gttataaact gtgaagtact cgtacagcag
     2581 agagtgttta ggaagcacct tttcgttagg cagattttta tcaaagttag tcatcctttc
     2641 gatgaaggac tgggcagagg cccccttatc cacgacttcc tcgaagttcc agggagtgat
     2701 ggtctcttct gatttgcgag tcatccacgc gaatctggaa tttccccggg cgagggggcc
     2761 tacatagtag ggtatccgaa atgtgaggat tttctcaatc ttttccctgt tatctttcaa
     2821 aaaggggtag aaatcctctt gccgcctgag gatagcgtgc agttcgccca ggtgaatctg
     2881 gtgggggatg cttccattgt cgaaagtgcg ctgtttgcgc aacagatctt ctctgttaag
     2941 ctttaccagc agctcctcgg tgccgtccat tttttccaag atgggcttaa taaatttgta
     3001 aaattcctcc tggcttgctc cgccgtcaat gtatccggcg tagccatttt tagactgatc
     3061 gaagaaaatt tccttgtact tctcaggcag ttgctgtctg acaagggcct tcagcaaagt
     3121 caagtcttgg tggtgctcat catagcgctt gatcatacta gcgctcagcg gagctttggt
     3181 gatctccgtg ttcactcgca gaatatcact cagcagaatg gcgtctgaca ggttctttgc
     3241 cgccaaaaaa aggtctgcgt actggtcgcc gatctgggcc agcagattgt cgagatcatc
     3301 atcgtaggtg tctttgctca gttgaagctt ggcatcttcg gccaggtcga agttagattt
     3361 aaagttgggg gtcagcccga gtgacagggc gataagatta ccaaacaggc cgttcttctt
     3421 ctccccaggg agctgtgcga tgaggttttc gagccgccgg gatttggaca gcctagcgct
     3481 caggattgct ttggcgtcaa ctccggatgc gttgatcggg ttctcttcga aaagctgatt
     3541 gtaagtctga accagttgga taaagagttt gtcgacatcg ctgttgtctg ggttcaggtc
     3601 cccctcgatg aggaagtgtc cccgaaattt gatcatatgc gccagcgcga gatagatcaa
     3661 ccgcaagtca gccttatcag tactgtctac aagcttcttc ctcagatgat atatggttgg
     3721 gtacttttca tggtacgcca cctcgtccac gatattgcca aagattgggt ggcgctcgtg
     3781 ctttttatcc tcctccacca aaaaggactc ctccagccta tggaagaaag agtcatccac
     3841 cttagccatc tcattactaa agatctcctg caggtagcag atccgattct ttctgcgggt
     3901 atatctgcgc cgtgctgttc ttttgagccg cgtggcttcg gccgtctccc cggagtcgaa
     3961 caggagggcg ccaatgaggt tcttctttat gctgtggcga tcggtattgc ccagaacttt
     4021 gaattttttg ctcggcacct tgtactcgtc cgtaatgacg gcccagccga cgctgtttgt
     4081 gccgatagcg agcccaatgg agtacttctt gtccat
//
