LOCUS       sequence-445557-        6728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and HIS3 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  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 6728)
  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-HIS (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..6728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        318..505
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             506..1168
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(1428..1883)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1565..1586)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1777..1796
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1962..2127)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2048..2072
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2272..2313)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        2366..3030
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2582..2699
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2994..3017
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3041..3060
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3041..3059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3083..3109
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3136..3325
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3397..3414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3568..4156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3648..3667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4327..5118,5119..5187))
                     /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     4950..4969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5188..5292)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5319..6661
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6282..6329)
                     /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     6702..6720
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccataaat tcccgtttta agagcttggt gagcgctagg agtcactgcc aggtatcgtt
      241 tgaacacggc attagtcagg gaagtcataa cacagtcctt tcccgcaatt ttctttttct
      301 attactcttg gcctcctcta gtacactcta tattttttta tgcctcggta atgattttca
      361 tttttttttt tccacctagc ggatgactct ttttttttct tagcgattgg cattatcaca
      421 taatgaatta tacattatat aaagtaatgt gatttcttcg aagaatatac taaaaaatga
      481 gcaggcaaga taaacgaagg caaagatgac agagcagaaa gccctagtaa agcgtattac
      541 aaatgaaacc aagattcaga ttgcgatctc tttaaagggt ggtcccctag cgatagagca
      601 ctcgatcttc ccagaaaaag aggcagaagc agtagcagaa caggccacac aatcgcaagt
      661 gattaacgtc cacacaggta tagggtttct ggaccatatg atacatgctc tggccaagca
      721 ttccggctgg tcgctaatcg ttgagtgcat tggtgactta cacatagacg accatcacac
      781 cactgaagac tgcgggattg ctctcggtca agcttttaaa gaggccctag gggccgtgcg
      841 tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt ccagagcggt
      901 ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa gggagaaagt
      961 aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag aggctagcag
     1021 aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta gtgagagtgc
     1081 gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta ccaacgatgt
     1141 tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg cagcatacga
     1201 tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta tacgaacagt
     1261 atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg aacgaggcgc
     1321 gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac ggatctatgc
     1381 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     1441 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1501 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     1561 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1621 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     1681 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1741 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1801 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1861 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     1921 cgatcggtgc gggcctcttc gctattacgc cagctgaatt ggagcgacct catgctatac
     1981 ctgagaaagc aacctgacct acaggaaaga gttactcaag aataagaatt ttcgttttaa
     2041 aacctaagag tcactttaaa atttgtatac acttattttt tttataactt atttaataat
     2101 aaaaatcata aatcataaga aattcgctta tttagaagtg tcaacaacgt atctaccaac
     2161 gatttgaccc ttttccatct tttcgtaaat ttctggcaag gtagacaagc cgacaacctt
     2221 gattggagac ttgaccaaac ctctggcgaa gaattgttaa ttaagagctc aggtagaatc
     2281 cagacccagc agagggttag ggataggctt accagatctc atcgatacta gtgcggccgc
     2341 cctttagtga gggttgaatt cgaattttca aaaattctta cttttttttt ggatggacgc
     2401 aaagaagttt aataatcata ttacatggca ttaccaccat atacatatcc atatacatat
     2461 ccatatctaa tcttacttat atgttgtgga aatgtaaaga gccccattat cttagcctaa
     2521 aaaaaccttc tctttggaac tttcagtaat acgcttaact gctcattgct atattgaagt
     2581 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt
     2641 cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga
     2701 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
     2761 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga
     2821 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat
     2881 taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc
     2941 ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac
     3001 ctctatactt taacgtcaag gagaaaaaac cccggatccg taatacgact cactataggg
     3061 cccgggcgtc gacatgctcg agtacccata cgatgttcca gattacgctt aagcttggta
     3121 ccgcggctag ctaagatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     3181 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     3241 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     3301 tgagaaggtt ttgggacgct cgaagatcca gctgcattaa tgaatcggcc aacgcgcggg
     3361 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3421 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3481 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3541 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3661 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3721 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3781 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3841 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3961 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4021 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4081 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4141 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4201 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4261 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4321 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4381 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4441 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4501 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4561 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4621 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4681 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4741 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4801 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4861 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4921 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4981 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5041 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5101 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5161 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5221 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5281 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     5341 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     5401 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     5461 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     5521 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     5581 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     5641 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     5701 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     5761 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     5821 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     5881 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     5941 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     6001 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     6061 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     6121 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     6181 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     6241 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     6301 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     6361 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     6421 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     6481 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     6541 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     6601 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     6661 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6721 cctttcgt
//
