LOCUS       sequence-346733-        9354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast low copy plasmid with dCas9 and sgRNA expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9354)
  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 9354)
  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_1_dCas9_sgRNA
FEATURES             Location/Qualifiers
     source          1..9354
                     /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"
     misc_RNA        5470..5545
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     gene            5712..7067
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             6056..6865
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      6870..7067
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    7072..7575
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(7619..8434)
                     /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(7619..8434)
                     /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     8426..8445
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8659..9247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9148..9167
                     /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 cctaactcga
     4921 gataaagcaa tcttgatgag gataatgatt tttttttgaa tatacataaa tactaccgtt
     4981 tttctgctag attttgtgat gacgtaaata agtacatatt actttttaag ccaagacaag
     5041 attaagcatt aactttaccc ttttctttct aagtttcaat attagttatc actgtttaaa
     5101 agttatggcg agaacgtcgg cggttaaaat atattaccct gaacggctgc caaaccagat
     5161 gtcaacacag ctacaacgat gtgcttcagt attacatttt ttgccttcaa cgccttgatt
     5221 gttctatttt tgctaataat aaatctattt catcggacta aaagtccatt agttgtaagc
     5281 ggatttagct cagttgggag agcgccagac tgaagaaaaa cttcggtcaa gtcatctgga
     5341 ggtcctgtgt tcgatccaca gaattcgcag atggccggca tggtcccagc ctcctcgctg
     5401 gcgccggctg ggcaacacct tcgggtggcg aatgggactc gctatcgtct tctcacgaag
     5461 acttactcag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     5521 tgaaaaagtg gcaccgagtc ggtgcttttt ttattttttg tcactattgt tatgtaaaat
     5581 gccacctctg acagtatgga acgcaaactt ctgtctagtg gatagctgag cagttacaga
     5641 gatgttacga accactagtg cactgcagta cagtttagct tgcctcgtcc ccgccgggtc
     5701 acccggccag cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct
     5761 caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat
     5821 ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctcc
     5881 agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc
     5941 ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc
     6001 cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa caaaaatggg
     6061 taaggaaaag actcacgttt cgaggccgcg attaaattcc aacatggatg ctgatttata
     6121 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     6181 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     6241 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     6301 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggcaa
     6361 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     6421 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     6481 tcgcgtattt cgtctggctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     6541 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     6601 gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     6661 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     6721 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     6781 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     6841 tttgatgctc gatgagtttt tctaaagtaa ctgacaataa aaagattctt gttttcaaga
     6901 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     6961 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     7021 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactggag tatcacgtgc
     7081 tataaaaata attataattt aaatttttta atataaatat ataaattaaa aatagaaagt
     7141 aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa gactctaggg
     7201 ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta ttaatgccga
     7261 attgtttcat cttgtctgtg tagaaaacca cacacgaaaa tcctgtgatt ttacatttta
     7321 cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat aaatatatat
     7381 gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt tcttcattcc
     7441 gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca taaaaataaa
     7501 taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc gcgtgtaagt
     7561 tacaggcaag cgatccgtcc cgagcggccg cgtgttacaa ccaattaacc aattctgatt
     7621 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     7681 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     7741 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     7801 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     7861 aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc
     7921 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     7981 cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     8041 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     8101 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     8161 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     8221 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     8281 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     8341 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     8401 tggagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     8461 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
     8521 gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc
     8581 agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     8641 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     8701 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     8761 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     8821 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     8881 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     8941 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     9001 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     9061 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     9121 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     9181 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     9241 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     9301 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgc
//
