LOCUS       sequence-346726-        9502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast low copy plasmid with dCas9, sgRNA expression cassette and 
            Mxi1 transcriptional repressor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9502)
  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 9502)
  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_3_dCas9_Mxi1_sgRNA
FEATURES             Location/Qualifiers
     source          1..9502
                     /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..5035
                     /codon_start=1
                     /product="transcriptional repressor domain from the mouse 
                     Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
                     /label=Mxi1
                     /translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
     CDS             5036..5056
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_RNA        5618..5693
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     gene            5860..7215
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             6204..7013
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      7018..7215
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    7220..7723
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(7767..8582)
                     /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(7767..8582)
                     /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     8574..8593
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8807..9395
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9296..9315
                     /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 ccatggaacg
     4921 tgtgagaatg attaatgtgc aaaggctgtt agaagccgca gagtttttag aaagaagaga
     4981 aagagaatgc gaacacgggt atgccagttc tttccctagc atgccctctc ccagaccgaa
     5041 aaagaaacgc aaagtttaac tcgagtggca taaagcaatc ttgatgagga taatgatttt
     5101 tttttgaata tacataaata ctaccgtttt tctgctagat tttgtgatga cgtaaataag
     5161 tacatattac tttttaagcc aagacaagat taagcattaa ctttaccctt ttctttctaa
     5221 gtttcaatat tagttatcac tgtttaaaag ttatggcgag aacgtcggcg gttaaaatat
     5281 attaccctga acggctgcca aaccagatgt caacacagct acaacgatgt gcttcagtat
     5341 tacatttttt gccttcaacg ccttgattgt tctatttttg ctaataataa atctatttca
     5401 tcggactaaa agtccattag ttgtaagcgg atttagctca gttgggagag cgccagactg
     5461 aagaaaaact tcggtcaagt catctggagg tcctgtgttc gatccacaga attcgcagat
     5521 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacaccttc gggtggcgaa
     5581 tgggactcgc tatcgtcttc tcacgaagac ttactcagtt ttagagctag aaatagcaag
     5641 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     5701 attttttgtc actattgtta tgtaaaatgc cacctctgac agtatggaac gcaaacttct
     5761 gtctagtgga tagctgagca gttacagaga tgttacgaac cactagtgca ctgcagtaca
     5821 gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc
     5881 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
     5941 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
     6001 cgaagcaaaa attacggctc ctcgctccag acctgcgagc agggaaacgc tcccctcaca
     6061 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
     6121 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
     6181 atcacatccg aacataaaca aaaatgggta aggaaaagac tcacgtttcg aggccgcgat
     6241 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     6301 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     6361 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     6421 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     6481 ggttactcac cactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
     6541 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     6601 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctggctcag gcgcaatcac
     6661 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     6721 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
     6781 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     6841 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     6901 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     6961 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taaagtaact
     7021 gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta
     7081 gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc
     7141 tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg
     7201 ctggtcgcta tactggagta tcacgtgcta taaaaataat tataatttaa attttttaat
     7261 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
     7321 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc
     7381 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaaaaccaca
     7441 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
     7501 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
     7561 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
     7621 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
     7681 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtcccg agcggccgcg
     7741 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     7801 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     7861 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     7921 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     7981 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
     8041 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     8101 accaaaccgt tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta
     8161 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     8221 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     8281 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     8341 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     8401 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     8461 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     8521 gcatccatgt tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc
     8581 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     8641 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata
     8701 aatcgaactt ttgctgagtt gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt
     8761 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     8821 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     8881 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     8941 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     9001 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     9061 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     9121 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     9181 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     9241 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     9301 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     9361 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     9421 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     9481 ctgtggataa ccgtgcggcc gc
//
