LOCUS       sequence-445564-        7051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and THR1 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7051)
  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 7051)
  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-THR (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..7051
                     /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"
     rep_origin      complement(1750..2205)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1887..1908)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2099..2118
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2284..2449)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2370..2394
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2594..2635)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        2688..3352
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2904..3021
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3316..3339
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3363..3382
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3363..3381
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3405..3431
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3458..3647
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3719..3736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3890..4478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3970..3989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4649..5440,5441..5509))
                     /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     5272..5291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5510..5614)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5641..6983
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6604..6651)
                     /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     7024..7042
                     /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 accataccag cttttcaatt caattcccct gcaggcttaa aattatcggt tctagcgatg
      241 ttagagtaca acctctgaac gccgttcctt agcatctttt gttattaatc agctctctgc
      301 tttgcattaa tttgaccacc tgccctgaaa cacctttctt ctagtccacc tgtgtaggct
      361 ttttcacctg aattgttcat tctcgatacg ccctcacgcc ggatgaaaaa tttgtgcaag
      421 agtcatcatc tcgaaaagat atggtgataa aaacaagtgt acttctaacc tgcctaatgg
      481 ttataacagt agcataaagt agatatacgg actacagaaa atggttcgtg ccttcaaaat
      541 taaagttcca gcttcttccg ccaatatcgg ccctggttat gatgttcttg gtgtcggtct
      601 ttctctattc ttggagttag atgtcaccat cgactccagc caagctcagg aaacaaacga
      661 cgatcccaac aactgcaagc tgtcttacac taaagaaagt gaaggctatt ctacggtccc
      721 attgcgttct gatgctaacc tgattaccag gactgcgtta tatgtgttgc gttgcaacaa
      781 tattagaaac ttcccctccg gaaccaaagt tcacgtctcc aacccaatcc cacttggccg
      841 tggattgggt tcctctggtg cagcagttgt ggcaggtgtt attttaggta acgaagtggc
      901 ccaattgggt ttctctaagc aacgtatgtt ggactactgt ttgatgattg aacgtcatcc
      961 agacaacata accgctgcta tgatgggagg cttttgcggt tcattcctaa gggacttgac
     1021 cccacaagag gtggaaagac gtgagattcc attggctgag gtgcttccag aaccttctgg
     1081 tggtgaagat accggtctgg ttcccccatt acctcccacc gatatcggta gacatgtcaa
     1141 ataccaatgg aaccccgcca ttaaatgtat tgcgatcatc ccacagttcg agttgtccac
     1201 cgccgactcc agaggcgttc ttccaaaagc ctacccaacc caggacttgg ttttcaatct
     1261 acaaagattg gccgtcttga ccacagcttt gaccatggac ccacctaatg ccgacttaat
     1321 ctaccctgct atgcaagatc gtgtccacca accttataga aagacattga tcccaggtct
     1381 cacggaaatc ttatcatgtg tcaccccatc cacataccct ggcctattgg gtatctgctt
     1441 gtcaggtgca ggcccaacta tcttggcttt ggccactgag aatttcgaag aaatctctca
     1501 agaaattatc aacaggttcg ccaaaaacgg catcaagtgc tcctggaaac tactggagcc
     1561 tgcctacgat ggtgcaagcg tcgaacagca atgaaatgtc ctttacccct agtatactgt
     1621 gttaaatact attgtagacc atatacatac tgtaattgac cgttaacggt tattatgctg
     1681 agtcttcttc cgttacctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     1741 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     1801 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     1861 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     1921 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     1981 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2041 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2101 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2161 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     2221 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgaa
     2281 ttggagcgac ctcatgctat acctgagaaa gcaacctgac ctacaggaaa gagttactca
     2341 agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat acacttattt
     2401 tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct tatttagaag
     2461 tgtcaacaac gtatctacca acgatttgac ccttttccat cttttcgtaa atttctggca
     2521 aggtagacaa gccgacaacc ttgattggag acttgaccaa acctctggcg aagaattgtt
     2581 aattaagagc tcaggtagaa tccagaccca gcagagggtt agggataggc ttaccagatc
     2641 tcatcgatac tagtgcggcc gccctttagt gagggttgaa ttcgaatttt caaaaattct
     2701 tacttttttt ttggatggac gcaaagaagt ttaataatca tattacatgg cattaccacc
     2761 atatacatat ccatatacat atccatatct aatcttactt atatgttgtg gaaatgtaaa
     2821 gagccccatt atcttagcct aaaaaaacct tctctttgga actttcagta atacgcttaa
     2881 ctgctcattg ctatattgaa gtacggatta gaagccgccg agcgggtgac agccctccga
     2941 aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt
     3001 gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg
     3061 aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga atcaaattaa
     3121 caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc
     3181 gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca taaccacttt
     3241 aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta ataaaagtat
     3301 caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatc
     3361 cgtaatacga ctcactatag ggcccgggcg tcgacatgct cgagtaccca tacgatgttc
     3421 cagattacgc ttaagcttgg taccgcggct agctaagatc cgctctaacc gaaaaggaag
     3481 gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa
     3541 gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta
     3601 acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc cagctgcatt
     3661 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3721 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3781 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3841 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3901 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3961 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4021 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4081 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4141 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4201 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4261 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4321 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4381 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4441 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4501 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4561 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4621 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4681 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4741 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4801 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4861 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4921 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4981 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5041 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5101 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5161 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5221 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5281 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5341 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5401 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5461 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5521 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5581 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5641 aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt
     5701 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt
     5761 taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa
     5821 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc
     5881 tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc
     5941 gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg
     6001 cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg
     6061 tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta
     6121 gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat
     6181 gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag
     6241 aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca
     6301 ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa
     6361 gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg
     6421 agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga
     6481 tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc
     6541 cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc
     6601 tctgaagttc ctatactttc tagagaatag gaacttcgga ataggaactt caaagcgttt
     6661 ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc acatacagct cactgttcac
     6721 gtcgcaccta tatctgcgtg ttgcctgtat atatatatac atgagaagaa cggcatagtg
     6781 cgtgtttatg cttaaatgcg tacttatatg cgtctattta tgtaggatga aaggtagtct
     6841 agtacctcct gtgatattat cccattccat gcggggtatc gtatgcttcc ttcagcacta
     6901 ccctttagct gttctatatg ctgccactcc tcaattggat tagtctcatc cttcaatgct
     6961 atcatttcct ttgatattgg atcatactaa gaaaccatta ttatcatgac attaacctat
     7021 aaaaataggc gtatcacgag gccctttcgt c
//
