LOCUS       sequence-445555-        6645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and URA3 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6645)
  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 6645)
  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-URA (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..6645
                     /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"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1878..2043)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1964..1988
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2188..2229)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        2282..2946
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2498..2615
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2910..2933
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     2957..2976
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2957..2975
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2999..3025
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3052..3241
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3313..3330)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3484..4072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3564..3583)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4243..5034,5035..5103))
                     /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     4866..4885
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5104..5208)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5235..6577
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6198..6245)
                     /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     6618..6636
                     /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 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tgaattggag cgacctcatg ctatacctga gaaagcaacc tgacctacag
     1921 gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt
     1981 gtatacactt atttttttta taacttattt aataataaaa atcataaatc ataagaaatt
     2041 cgcttattta gaagtgtcaa caacgtatct accaacgatt tgaccctttt ccatcttttc
     2101 gtaaatttct ggcaaggtag acaagccgac aaccttgatt ggagacttga ccaaacctct
     2161 ggcgaagaat tgttaattaa gagctcaggt agaatccaga cccagcagag ggttagggat
     2221 aggcttacca gatctcatcg atactagtgc ggccgccctt tagtgagggt tgaattcgaa
     2281 ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata atcatattac
     2341 atggcattac caccatatac atatccatat acatatccat atctaatctt acttatatgt
     2401 tgtggaaatg taaagagccc cattatctta gcctaaaaaa accttctctt tggaactttc
     2461 agtaatacgc ttaactgctc attgctatat tgaagtacgg attagaagcc gccgagcggg
     2521 tgacagccct ccgaaggaag actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc
     2581 tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag
     2641 cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatga
     2701 acgaatcaaa ttaacaacca taggatgata atgcgattag ttttttagcc ttatttctgg
     2761 ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa atgcaaaaac
     2821 tgcataacca ctttaactaa tactttcaac attttcggtt tgtattactt cttattcaaa
     2881 tgtaataaaa gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga
     2941 aaaaaccccg gatccgtaat acgactcact atagggcccg ggcgtcgaca tgctcgagta
     3001 cccatacgat gttccagatt acgcttaagc ttggtaccgc ggctagctaa gatccgctct
     3061 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt
     3121 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg
     3181 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     3241 gatccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3301 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3361 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3421 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3481 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3541 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3601 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3661 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3721 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3781 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3841 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3901 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3961 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4021 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4081 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4141 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4201 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4261 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4321 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4381 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4441 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4501 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4561 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4621 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4681 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4741 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4801 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4861 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4921 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4981 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5041 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5101 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5161 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5221 aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga
     5281 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     5341 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa
     5401 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     5461 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa
     5521 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg
     5581 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa
     5641 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg
     5701 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta
     5761 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat
     5821 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat
     5881 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa
     5941 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag
     6001 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat
     6061 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt
     6121 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg
     6181 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga
     6241 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac
     6301 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga
     6361 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg
     6421 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc
     6481 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct
     6541 catccttcaa tgctatcatt tcctttgata ttggatcata ctaagaaacc attattatca
     6601 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
