LOCUS       sequence-346734-        9532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast low copy plasmid with dCas9, sgRNA expression cassette and 
            VP64 activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9532)
  AUTHORS   Camara E, Lenitz I, Nygard Y
  TITLE     A CRISPR activation and interference toolkit for industrial 
            Saccharomyces cerevisiae strain KE6-12.
  JOURNAL   Sci Rep. 2020 Sep 3;10(1):14605. doi: 10.1038/s41598-020-71648-w.
  PUBMED    32884066
REFERENCE   2  (bases 1 to 9532)
  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: "Sci Rep."; 
            date: "2020-09-3"; volume: "10(1):14605. doi"; pages: " 
            10.1038/s41598-020-71648-w"
COMMENT     Sequence Label: EC2_2_dCas9_VP64_sgRNA
FEATURES             Location/Qualifiers
     source          1..9532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        322..735
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             758..778
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4886..4906
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4913..5062
                     /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             5066..5086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_RNA        5648..5723
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     gene            5890..7245
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             6234..7043
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      7048..7245
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    7250..7753
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(7797..8612)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7797..8612)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     8604..8623
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8837..9425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9326..9345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgaattc gcatctagac tgaactggcc gataattgca gacgaacgcc ttgccaacag
       61 ggagttcttc agagacatgg aggctcaaaa cgaaattatt gacagcctag acatcaatag
      121 tcatacaaca gaaagcgacc acccaacttt ggctgataat agcgtataaa caatgcatac
      181 tttgtacgtt caaaatacaa tgcagtagat atatttatgc atattacata taatacatat
      241 cacataggaa gcaacaggcg cgttggactt ttaattttcg aggaccgcga atccttacat
      301 cacacccaat cccccacaag tgatccccca cacaccatag cttcaaaatg tttctactcc
      361 ttttttactc ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc
      421 aagcacagca tactaaattt cccctctttc ttcctctagg gtgtcgttaa ttacccgtac
      481 taaaggtttg gaaaagaaaa aagacaccgc ctcgtttctt tttcttcgtc gaaaaaggca
      541 ataaaaattt ttatcacgtt tctttttctt gaaaattttt ttttttgatt tttttctctt
      601 tcgatgacct cccattgata tttaagttaa taaacggtca tcaatttctc aagtttcagt
      661 ttcatttttc ttgttctatt acaacttttt ttacttcttg ctcattagaa agaaagcata
      721 gcaatctaat ctaagtttta attacaaaag atctatgcca aagaagaaga gaaaggtagg
      781 cagcgacaag aagtattcta tcggactggc catcgggact aatagcgtcg ggtgggccgt
      841 gatcactgac gagtacaagg tgccctctaa gaagttcaag gtgctcggga acaccgaccg
      901 gcattccatc aagaaaaatc tgatcggagc tctcctcttt gattcagggg aaaccgctga
      961 agcaacccgc ctcaagcgga ctgctagacg gcggtacacc aggaggaaga accggatttg
     1021 ttaccttcaa gagatattct ccaacgaaat ggcaaaggtc gacgacagct tcttccatag
     1081 gctggaagaa tcattcctcg tggaagagga taagaagcat gaacggcatc ccatcttcgg
     1141 taatatcgtc gacgaggtgg cctatcacga gaaataccca accatctacc atcttcgcaa
     1201 aaagctggtg gactcaaccg acaaggcaga cctccggctt atctacctgg ccctggccca
     1261 catgatcaag ttcagaggcc acttcctgat cgagggcgac ctcaatcctg acaatagcga
     1321 tgtggataaa ctgttcatcc agctggtgca gacttacaac cagctctttg aagagaaccc
     1381 catcaatgca agcggagtcg atgccaaggc cattctgtca gcccggctgt caaagagccg
     1441 cagacttgag aatcttatcg ctcagctgcc gggtgaaaag aaaaatggac tgttcgggaa
     1501 cctgattgct ctttcacttg ggctgactcc caatttcaag tctaatttcg acctggcaga
     1561 ggatgccaag ctgcaactgt ccaaggacac ctatgatgac gatctcgaca acctcctggc
     1621 ccagatcggt gaccaatacg ccgacctttt ccttgctgct aagaatcttt ctgacgccat
     1681 cctgctgtct gacattctcc gcgtgaacac tgaaatcacc aaggcccctc tttcagcttc
     1741 aatgattaag cggtatgatg agcaccacca ggacctgacc ctgcttaagg cactcgtccg
     1801 gcagcagctt ccggagaagt acaaggaaat cttctttgac cagtcaaaga atggatacgc
     1861 cggctacatc gacggaggtg cctcccaaga ggaattttat aagtttatca aacctatcct
     1921 tgagaagatg gacggcaccg aagagctcct cgtgaaactg aatcgggagg atctgctgcg
     1981 gaagcagcgc actttcgaca atgggagcat tccccaccag atccatcttg gggagcttca
     2041 cgccatcctt cggcgccaag aggacttcta cccctttctt aaggacaaca gggagaagat
     2101 tgagaaaatt ctcactttcc gcatccccta ctacgtggga cccctcgcca gaggaaatag
     2161 ccggtttgct tggatgacca gaaagtcaga agaaactatc actccctgga acttcgaaga
     2221 ggtggtggac aagggagcca gcgctcagtc attcatcgaa cggatgacta acttcgataa
     2281 gaacctcccc aatgagaagg tcctgccgaa acattccctg ctctacgagt actttaccgt
     2341 gtacaacgag ctgaccaagg tgaaatatgt caccgaaggg atgaggaagc ccgcattcct
     2401 gtcaggcgaa caaaagaagg caattgtgga ccttctgttc aagaccaata gaaaggtgac
     2461 cgtgaagcag ctgaaggagg actatttcaa gaaaattgaa tgcttcgact ctgtggagat
     2521 tagcggggtc gaagatcggt tcaacgcaag cctgggtacc taccatgatc tgcttaagat
     2581 catcaaggac aaggattttc tggacaatga ggagaaagag gacatccttg aggacattgt
     2641 cctgactctc actctgttcg aggaccggga aatgatcgag gagaggctta agacctacgc
     2701 ccatctgttc gacgataaag tgatgaagca acttaaacgg agaagatata ccggatgggg
     2761 acgccttagc cgcaaactca tcaacggaat ccgggacaaa cagagcggaa agaccattct
     2821 tgatttcctt aagagcgacg gattcgctaa tcgcaacttc atgcaactta tccatgatga
     2881 ttccctgacc tttaaggagg acatccagaa ggcccaagtg tctggacaag gtgactcact
     2941 gcacgagcat atcgcaaatc tggctggttc acccgctatt aagaagggta ttctccagac
     3001 cgtgaaagtc gtggacgagc tggtcaaggt gatgggtcgc cataaaccag agaacattgt
     3061 catcgagatg gccagggaaa accagactac ccagaaggga cagaagaaca gcagggagcg
     3121 gatgaaaaga attgaggaag ggattaagga gctcgggtca cagatcctta aagagcaccc
     3181 ggtggaaaac acccagcttc agaatgagaa gctctatctg tactaccttc aaaatggacg
     3241 cgatatgtat gtggaccaag agcttgatat caacaggctc tcagactacg acgtggacgc
     3301 catcgtccct cagagcttcc tcaaagacga ctcaattgac aataaggtgc tgactcgctc
     3361 agacaagaac cggggaaagt cagataacgt gccctcagag gaagtcgtga aaaagatgaa
     3421 gaactattgg cgccagcttc tgaacgcaaa gctgatcact cagcggaagt tcgacaatct
     3481 cactaaggct gagaggggcg gactgagcga actggacaaa gcaggattca ttaaacggca
     3541 acttgtggag actcggcaga ttactaaaca tgtagcccaa atccttgact cacgcatgaa
     3601 taccaagtac gacgaaaacg acaaacttat ccgcgaggtg aaggtgatta ccctgaagtc
     3661 caagctggtc agcgatttca gaaaggactt tcaattctac aaagtgcggg agatcaataa
     3721 ctatcatcat gctcatgacg catatctgaa tgccgtggtg ggaaccgccc tgatcaagaa
     3781 gtacccaaag ctggaaagcg agttcgtgta cggagactac aaggtctacg acgtgcgcaa
     3841 gatgattgcc aaatctgagc aggagatcgg aaaggccacc gcaaagtact tcttctacag
     3901 caacatcatg aatttcttca agaccgaaat cacccttgca aacggtgaga tccggaagag
     3961 gccgctcatc gagactaatg gggagactgg cgaaatcgtg tgggacaagg gcagagattt
     4021 cgctaccgtg cgcaaagtgc tttctatgcc tcaagtgaac atcgtgaaga aaaccgaggt
     4081 gcaaaccgga ggcttttcta aggaatcaat cctccccaag cgcaactccg acaagctcat
     4141 tgcaaggaag aaggattggg accctaagaa gtacggcgga ttcgattcac caactgtggc
     4201 ttattctgtc ctggtcgtgg ctaaggtgga aaaaggaaag tctaagaagc tcaagagcgt
     4261 gaaggaactg ctgggtatca ccattatgga gcgcagctcc ttcgagaaga acccaattga
     4321 ctttctcgaa gccaaaggtt acaaggaagt caagaaggac cttatcatca agctcccaaa
     4381 gtatagcctg ttcgaactgg agaatgggcg gaagcggatg ctcgcctccg ctggcgaact
     4441 tcagaagggt aatgagctgg ctctcccctc caagtacgtg aatttcctct accttgcaag
     4501 ccattacgag aagctgaagg ggagccccga ggacaacgag caaaagcaac tgtttgtgga
     4561 gcagcataag cattatctgg acgagatcat tgagcagatt tccgagtttt ctaaacgcgt
     4621 cattctcgct gatgccaacc tcgataaagt ccttagcgca tacaataagc acagagacaa
     4681 accaattcgg gagcaggctg agaatatcat ccacctgttc accctcacca atcttggtgc
     4741 ccctgccgca ttcaagtact tcgacaccac catcgaccgg aaacgctata cctccaccaa
     4801 agaagtgctg gacgccaccc tcatccacca gagcatcacc ggactttacg aaactcggat
     4861 tgacctctca cagctcggag gggatcccaa gaaaaagcgc aaggtaggat ccgacgcatt
     4921 ggacgatttt gatctggata tgctgggaag tgacgccctc gatgattttg accttgacat
     4981 gcttggttcg gatgcccttg atgactttga cctcgacatg ctcggcagtg acgcccttga
     5041 tgatttcgac ctggacatgc tgattccgaa aaagaaacgc aaagtttaac tcgagtggca
     5101 taaagcaatc ttgatgagga taatgatttt tttttgaata tacataaata ctaccgtttt
     5161 tctgctagat tttgtgatga cgtaaataag tacatattac tttttaagcc aagacaagat
     5221 taagcattaa ctttaccctt ttctttctaa gtttcaatat tagttatcac tgtttaaaag
     5281 ttatggcgag aacgtcggcg gttaaaatat attaccctga acggctgcca aaccagatgt
     5341 caacacagct acaacgatgt gcttcagtat tacatttttt gccttcaacg ccttgattgt
     5401 tctatttttg ctaataataa atctatttca tcggactaaa agtccattag ttgtaagcgg
     5461 atttagctca gttgggagag cgccagactg aagaaaaact tcggtcaagt catctggagg
     5521 tcctgtgttc gatccacaga attcgcagat ggccggcatg gtcccagcct cctcgctggc
     5581 gccggctggg caacaccttc gggtggcgaa tgggactcgc tatcgtcttc tcacgaagac
     5641 ttactcagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     5701 aaaaagtggc accgagtcgg tgcttttttt attttttgtc actattgtta tgtaaaatgc
     5761 cacctctgac agtatggaac gcaaacttct gtctagtgga tagctgagca gttacagaga
     5821 tgttacgaac cactagtgca ctgcagtaca gtttagcttg cctcgtcccc gccgggtcac
     5881 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
     5941 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
     6001 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctccag
     6061 acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc
     6121 ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct
     6181 tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca aaaatgggta
     6241 aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg
     6301 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     6361 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     6421 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     6481 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa
     6541 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     6601 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     6661 gcgtatttcg tctggctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     6721 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc
     6781 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     6841 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     6901 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     6961 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     7021 tgatgctcga tgagtttttc taaagtaact gacaataaaa agattcttgt tttcaagaac
     7081 ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg
     7141 atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa
     7201 gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactggagta tcacgtgcta
     7261 taaaaataat tataatttaa attttttaat ataaatatat aaattaaaaa tagaaagtaa
     7321 aaaaagaaat taaagaaaaa atagtttttg ttttccgaag atgtaaaaga ctctaggggg
     7381 atcgccaaca aatactacct tttatcttgc tcttcctgct ctcaggtatt aatgccgaat
     7441 tgtttcatct tgtctgtgta gaaaaccaca cacgaaaatc ctgtgatttt acattttact
     7501 tatcgttaat cgaatgtata tctatttaat ctgcttttct tgtctaataa atatatatgt
     7561 aaagtacgct ttttgttgaa attttttaaa cctttgttta tttttttttc ttcattccgt
     7621 aactcttcta ccttctttat ttactttcta aaatccaaat acaaaacata aaaataaata
     7681 aacacagagt aaattcccaa attattccat cattaaaaga tacgaggcgc gtgtaagtta
     7741 caggcaagcg atccgtcccg agcggccgcg tgttacaacc aattaaccaa ttctgattag
     7801 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     7861 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     7921 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     7981 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     8041 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     8101 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     8161 tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     8221 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     8281 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     8341 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     8401 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     8461 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     8521 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg
     8581 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     8641 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     8701 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag
     8761 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     8821 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     8881 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     8941 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     9001 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     9061 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     9121 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     9181 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     9241 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     9301 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     9361 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     9421 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     9481 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gc
//
