LOCUS       sequence-445556-        7768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and LEU2 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7768)
  AUTHORS   Dong G, Nakai T, Matsuzaki T
  TITLE     A novel plasmid-based experimental system in Saccharomyces 
            cerevisiae that enables the introduction of 10 different plasmids 
            into cells.
  JOURNAL   FEBS Open Bio. 2024 Oct 10. doi: 10.1002/2211-5463.13893.
  PUBMED    39387713
REFERENCE   2  (bases 1 to 7768)
  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: "FEBS Open 
            Bio. 2024 Oct 10. doi: 10.1002/2211-5463.13893."
COMMENT     Sequence Label: pESC-LEU (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..7768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(664..1758)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1759..2166)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2467..2922)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2604..2625)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2816..2835
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3001..3166)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3087..3111
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3311..3352)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        3405..4069
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3621..3738
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4033..4056
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     4080..4099
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4080..4098
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4122..4148
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4175..4364
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4436..4453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4607..5195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4687..4706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5366..6157,6158..6226))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5989..6008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6227..6331)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6358..7700
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7321..7368)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7741..7759
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgtt aaggccgttt ctgacagagt aaaattcttg agggaacttt
      241 caccattatg ggaaatggtt caagaaggta ttgacttaaa ctccatcaaa tggtcaggtc
      301 attgagtgtt ttttatttgt tgtatttttt tttttttaga gaaaatcctc caatatataa
      361 attaggaatc atagtttcat gattttctgt tacacctaac tttttgtgtg gtgccctcct
      421 ccttgtcaat attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta
      481 tcaatttgct tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg
      541 tagattgcgt atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc
      601 gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc
      661 tttttaagca aggattttct taacttcttc ggcgacagca tcaccgactt cggtggtact
      721 gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc
      781 ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga
      841 caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca
      901 tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg
      961 caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat
     1021 gttgctggtg attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc
     1081 agaatcaatc aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc
     1141 cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat
     1201 aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac
     1261 ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct
     1321 cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc
     1381 aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct
     1441 taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac
     1501 actaccggta ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt
     1561 ggaggcttcc agcgcctcat ctggaagtgg aacacctgta gcatcgatag cagcaccacc
     1621 aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag
     1681 aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat
     1741 cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat atattgctga
     1801 aatgtaaaag gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa
     1861 caataggtcc ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg
     1921 aacgcttctc tattctatat gaaaagccgg ttccggcgct ctcacctttc ctttttctcc
     1981 caatttttca gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt
     2041 ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga
     2101 aaaaaataat ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc
     2161 acagttaact gcggtcaaga tatttcttga atcaggcgcc ttagaccgct cggccaaaca
     2221 accaattact tgttgagaaa tagagtataa ttatcctata aatataacgt ttttgaacac
     2281 acatgaacaa ggaagtacag gacaattgat tttgaagaga atgtggattt tgatgtaatt
     2341 gttgggattc catttttaat aaggcaataa tattaggtat gtagatatac tagaagttct
     2401 cctcgaccgg tcgatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     2461 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     2941 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgaattg
     3001 gagcgacctc atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga
     3061 ataagaattt tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt
     3121 ttataactta tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt
     3181 caacaacgta tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg
     3241 tagacaagcc gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat
     3301 taagagctca ggtagaatcc agacccagca gagggttagg gataggctta ccagatctca
     3361 tcgatactag tgcggccgcc ctttagtgag ggttgaattc gaattttcaa aaattcttac
     3421 tttttttttg gatggacgca aagaagttta ataatcatat tacatggcat taccaccata
     3481 tacatatcca tatacatatc catatctaat cttacttata tgttgtggaa atgtaaagag
     3541 ccccattatc ttagcctaaa aaaaccttct ctttggaact ttcagtaata cgcttaactg
     3601 ctcattgcta tattgaagta cggattagaa gccgccgagc gggtgacagc cctccgaagg
     3661 aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc
     3721 tcgcgccgca ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag
     3781 aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa tgaacgaatc aaattaacaa
     3841 ccataggatg ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa
     3901 gcgatgattt ttgatctatt aacagatata taaatgcaaa aactgcataa ccactttaac
     3961 taatactttc aacattttcg gtttgtatta cttcttattc aaatgtaata aaagtatcaa
     4021 caaaaaattg ttaatatacc tctatacttt aacgtcaagg agaaaaaacc ccggatccgt
     4081 aatacgactc actatagggc ccgggcgtcg acatgctcga gtacccatac gatgttccag
     4141 attacgctta agcttggtac cgcggctagc taagatccgc tctaaccgaa aaggaaggag
     4201 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     4261 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     4321 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaagatccag ctgcattaat
     4381 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5041 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5221 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5341 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5401 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5461 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5521 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5581 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5641 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5701 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5761 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5821 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5881 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5941 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6001 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6061 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6121 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6181 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6241 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6301 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac
     6361 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     6421 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     6481 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     6541 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     6601 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     6661 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     6721 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     6781 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     6841 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     6901 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     6961 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     7021 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     7081 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     7141 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     7201 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     7261 tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct
     7321 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg
     7381 aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc
     7441 gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt
     7501 gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt
     7561 acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc
     7621 tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc
     7681 atttcctttg atattggatc atactaagaa accattatta tcatgacatt aacctataaa
     7741 aataggcgta tcacgaggcc ctttcgtc
//
