LOCUS       sequence-127064-        4695 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 4695)
  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 4695)
  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..4695
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(7..156)
                     /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             complement(join(205..1719,1720..1722,1723..1788,1789..1794,
                     1795..4278,4279..4281,4282..4308))
                     /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(205..1788,1789..1791,1792..4278,4279..4281,
                     4282..4308))
                     /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(205..4308)
                     /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"
     protein_bind    complement(4311..4335)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4351..4692)
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
ORIGIN
        1 ctaggaaagc atgtctaagt cgaaatcgtc cagagcgtct gatccaagca tatccaggtc
       61 aaaatcatct aaggcgtcac ttccaagcat gtcgaggtca aagtcatcta gcgcatcaga
      121 acccagcata tccaagtcga agtcgtccaa agcgtcagaa cctccaccgt cgactccaga
      181 tctaccagaa ccgtcagccc tgctgtctcc accgagctga gagaggtcga ttcttgtttc
      241 atagagcccc gtaattgact gatgaatcag tgtggcgtcc aggacctcct ttgtagaggt
      301 gtaccgcttt ctgtctatgg tggtgtcgaa gtacttgaag gctgcaggcg cgcccaagtt
      361 ggtcagagta aacaagtgga taatgttttc tgcctgctcc ctgatgggct tatccctgtg
      421 cttattgtaa gcagaaagca ccttatcgag gttagcgtcg gcgaggatca ctcttttgga
      481 gaattcgctt atttgctcga tgatctcatc aaggtagtgt ttgtgttgtt ccacgaacag
      541 ctgcttctgc tcattatctt cgggagaccc tttgagcttt tcatagtggc tggccagata
      601 caagaaatta acgtatttag agggcagtgc cagctcgtta cctttctgca gctcgcccgc
      661 actagcgagc attcgtttcc ggccgttttc aagctcaaag agagagtact tgggaagctt
      721 aatgatgagg tcttttttga cctctttata tcctttcgcc tcgagaaagt cgatggggtt
      781 tttttcgaag cttgatcgct ccatgattgt gatgcccagc agttccttga cgcttttgag
      841 ttttttagac ttccctttct ccactttggc cacaaccagt acactgtaag cgactgtagg
      901 agaatcgaat ccgccgtatt tcttggggtc ccaatctttt ttgcgtgcga tcagcttgtc
      961 gctgttcctt ttcgggagga tactttcctt ggagaagcct ccggtctgta cttcggtctt
     1021 tttaacgatg ttcacctgcg gcatggacag gaccttccgg actgtcgcga aatccctacc
     1081 cttgtcccac acgatttctc ctgtttctcc gtttgtttcg ataagtggtc gcttccgaat
     1141 ctctccattg gccagtgtaa tctcggtctt gaaaaaattc ataatattgc tgtaaaagaa
     1201 gtacttagcg gtggccttgc ctatttcctg ctcagacttt gcgatcattt tcctaacatc
     1261 gtacacttta tagtctccgt aaacaaattc agattcaagc ttgggatatt ttttgataag
     1321 tgcagtgcct accactgcat tcaggtaggc atcatgcgca tggtggtaat tgttgatctc
     1381 tctcacctta taaaactgaa agtcctttct gaaatctgag accagcttag acttcagagt
     1441 aataactttc acctctcgaa tcagtttgtc attttcatcg tacttggtgt tcatgcgtga
     1501 atcgagaatt tgggccacgt gcttggtgat ctggcgtgtc tcaacaagct gccttttgat
     1561 gaagccggct ttatccaact cagacaggcc acctcgttca gccttagtca gattatcgaa
     1621 cttccgttgt gtgatcagtt tggcgttcag cagctgccgc caataatttt tcattttctt
     1681 gacaacttct tctgagggga cgttatcact cttccctcta tttttatcgg atcttgtcaa
     1741 cactttatta tcaatagaat catctttgag aaaagactgg ggcacgatag catccacgtc
     1801 gtagtcggag agccgattga tgtccagttc ctgatccacg tacatgtccc tgccgttctg
     1861 caggtagtac aggtagagct tctcattctg aagctgggtg ttttcaactg ggtgttcctt
     1921 aaggatttgg gaccccagtt cttttatacc ctcttcaatc ctcttcatcc tttccctact
     1981 gttcttctgt cccttctggg tagtttggtt ctctcgggcc atctcgataa cgatattctc
     2041 gggcttatgc cttcccatta ctttgacgag ttcatccacg accttaacgg tctgcagtat
     2101 tccctttttg atagctgggc tacctgcaag attagcgatg tgctcgtgaa gactgtcccc
     2161 ctggccagaa acttgtgctt tctggatgtc ctccttaaag gtgagagagt catcatggat
     2221 caactgcatg aagttccggt tggcaaatcc atcggactta agaaaatcca ggattgtctt
     2281 tccactctgc ttgtctcgga tcccattgat cagttttctt gacagccgcc cccatcctgt
     2341 atatcggcgc ctcttgagct gtttcatgac tttgtcgtcg aagagatgag cgtaagtttt
     2401 caagcgttct tcaatcatct ccctatcttc aaacaacgta agggtgagga caatgtcctc
     2461 aagaatgtcc tcgttctcct cattgtccag gaagtccttg tctttaatga ttttcaggag
     2521 atcgtgatac gttcccaggg atgcgttgaa gcgatcctcc actccgctga tttcaacaga
     2581 gtcgaaacat tcaatctttt tgaaatagtc ttctttgagc tgtttcacgg taactttccg
     2641 gttcgtcttg aagaggaggt ccacgatagc tttcttctgc tctccagaca ggaatgctgg
     2701 ctttctcatc ccttctgtga cgtatttgac cttggtgagc tcgttataaa ctgtgaagta
     2761 ctcgtacagc agagagtgtt taggaagcac cttttcgtta ggcagatttt tatcaaagtt
     2821 agtcatcctt tcgatgaagg actgggcaga ggccccctta tccacgactt cctcgaagtt
     2881 ccagggagtg atggtctctt ctgatttgcg agtcatccac gcgaatctgg aatttccccg
     2941 ggcgaggggg cctacatagt agggtatccg aaatgtgagg attttctcaa tcttttccct
     3001 gttatctttc aaaaaggggt agaaatcctc ttgccgcctg aggatagcgt gcagttcgcc
     3061 caggtgaatc tggtggggga tgcttccatt gtcgaaagtg cgctgtttgc gcaacagatc
     3121 ttctctgtta agctttacca gcagctcctc ggtgccgtcc attttttcca agatgggctt
     3181 aataaatttg taaaattcct cctggcttgc tccgccgtca atgtatccgg cgtagccatt
     3241 tttagactga tcgaagaaaa tttccttgta cttctcaggc agttgctgtc tgacaagggc
     3301 cttcagcaaa gtcaagtctt ggtggtgctc atcatagcgc ttgatcatac tagcgctcag
     3361 cggagctttg gtgatctccg tgttcactcg cagaatatca ctcagcagaa tggcgtctga
     3421 caggttcttt gccgccaaaa aaaggtctgc gtactggtcg ccgatctggg ccagcagatt
     3481 gtcgagatca tcatcgtagg tgtctttgct cagttgaagc ttggcatctt cggccaggtc
     3541 gaagttagat ttaaagttgg gggtcagccc gagtgacagg gcgataagat taccaaacag
     3601 gccgttcttc ttctccccag ggagctgtgc gatgaggttt tcgagccgcc gggatttgga
     3661 cagcctagcg ctcaggattg ctttggcgtc aactccggat gcgttgatcg ggttctcttc
     3721 gaaaagctga ttgtaagtct gaaccagttg gataaagagt ttgtcgacat cgctgttgtc
     3781 tgggttcagg tccccctcga tgaggaagtg tccccgaaat ttgatcatat gcgccagcgc
     3841 gagatagatc aaccgcaagt cagccttatc agtactgtct acaagcttct tcctcagatg
     3901 atatatggtt gggtactttt catggtacgc cacctcgtcc acgatattgc caaagattgg
     3961 gtggcgctcg tgctttttat cctcctccac caaaaaggac tcctccagcc tatggaagaa
     4021 agagtcatcc accttagcca tctcattact aaagatctcc tgcaggtagc agatccgatt
     4081 ctttctgcgg gtatatctgc gccgtgctgt tcttttgagc cgcgtggctt cggccgtctc
     4141 cccggagtcg aacaggaggg cgccaatgag gttcttcttt atgctgtggc gatcggtatt
     4201 gcccagaact ttgaattttt tgctcggcac cttgtactcg tccgtaatga cggcccagcc
     4261 gacgctgttt gtgccgatag cgagcccaat ggagtacttc ttgtccatag agcctgcttt
     4321 tttgtacaaa cttgtttgat tcgacccacc ctcttttttt gggtttggtg gggtatcttc
     4381 atcatcgaat agatagttat atacatcatc cattgtagtg gtattaaaca tccctgtagt
     4441 gattccaaac gcgttatacg cagtttggtc cgtccaacca ggtgacagtg gttttgaatt
     4501 attaccatca tcaattttac tagccgtgat ttcattattc atgaagttat catgaacgtt
     4561 agaggaggca attggttgtg aaagcgcttg agaatttgtt tgagttgtta tgaggttcgg
     4621 accgttgcta ctgttagtga aagtgaagga caatgagcta tcagcaatat tcccactttg
     4681 attaaaattg gcgcc
//
