LOCUS       sequence-442027-        6551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and TRP1 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  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 6551)
  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-TRP (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..6551
                     /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        191..472
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             473..1147
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1250..1705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1599..1618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1784..1949)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1870..1894
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2094..2135)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        2188..2852
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2404..2521
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2816..2839
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     2863..2882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2863..2881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2905..2931
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      2958..3147
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3219..3236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3390..3978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3470..3489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4149..4940,4941..5009))
                     /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     4772..4791
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5010..5114)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5141..6483
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6104..6151)
                     /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     6524..6542
                     /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 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc
      241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta
      301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg
      361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
      421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
      481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
      541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
      601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
      661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
      721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
      781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
      841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
      901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
      961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     1201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1501 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1561 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1621 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1681 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1741 ggcgatcggt gcgggcctct tcgctattac gccagctgaa ttggagcgac ctcatgctat
     1801 acctgagaaa gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt
     1861 aaaacctaag agtcacttta aaatttgtat acacttattt tttttataac ttatttaata
     1921 ataaaaatca taaatcataa gaaattcgct tatttagaag tgtcaacaac gtatctacca
     1981 acgatttgac ccttttccat cttttcgtaa atttctggca aggtagacaa gccgacaacc
     2041 ttgattggag acttgaccaa acctctggcg aagaattgtt aattaagagc tcaggtagaa
     2101 tccagaccca gcagagggtt agggataggc ttaccagatc tcatcgatac tagtgcggcc
     2161 gccctttagt gagggttgaa ttcgaatttt caaaaattct tacttttttt ttggatggac
     2221 gcaaagaagt ttaataatca tattacatgg cattaccacc atatacatat ccatatacat
     2281 atccatatct aatcttactt atatgttgtg gaaatgtaaa gagccccatt atcttagcct
     2341 aaaaaaacct tctctttgga actttcagta atacgcttaa ctgctcattg ctatattgaa
     2401 gtacggatta gaagccgccg agcgggtgac agccctccga aggaagactc tcctccgtgc
     2461 gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt gcctcgcgcc gcactgctcc
     2521 gaacaataaa gattctacaa tactagcttt tatggttatg aagaggaaaa attggcagta
     2581 acctggcccc acaaaccttc aaatgaacga atcaaattaa caaccatagg atgataatgc
     2641 gattagtttt ttagccttat ttctggggta attaatcagc gaagcgatga tttttgatct
     2701 attaacagat atataaatgc aaaaactgca taaccacttt aactaatact ttcaacattt
     2761 tcggtttgta ttacttctta ttcaaatgta ataaaagtat caacaaaaaa ttgttaatat
     2821 acctctatac tttaacgtca aggagaaaaa accccggatc cgtaatacga ctcactatag
     2881 ggcccgggcg tcgacatgct cgagtaccca tacgatgttc cagattacgc ttaagcttgg
     2941 taccgcggct agctaagatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     3001 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     3061 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     3121 cttgagaagg ttttgggacg ctcgaagatc cagctgcatt aatgaatcgg ccaacgcgcg
     3181 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3241 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3301 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3361 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3421 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3481 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3541 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3601 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3661 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3721 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3781 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3841 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3901 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3961 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4021 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4081 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4141 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4201 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4261 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4321 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4381 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4441 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4501 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4561 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4621 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4681 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4741 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4801 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4861 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4921 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4981 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5041 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5101 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aacgaagcat ctgtgcttca
     5161 ttttgtagaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg
     5221 catttttaca gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc
     5281 ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg
     5341 agctgcattt ttacagaaca gaaatgcaac gcgagagcgc tattttacca acaaagaatc
     5401 tatacttctt ttttgttcta caaaaatgca tcccgagagc gctatttttc taacaaagca
     5461 tcttagatta ctttttttct cctttgtgcg ctctataatg cagtctcttg ataacttttt
     5521 gcactgtagg tccgttaagg ttagaagaag gctactttgg tgtctatttt ctcttccata
     5581 aaaaaagcct gactccactt cccgcgttta ctgattacta gcgaagctgc gggtgcattt
     5641 tttcaagata aaggcatccc cgattatatt ctataccgat gtggattgcg catactttgt
     5701 gaacagaaag tgatagcgtt gatgattctt cattggtcag aaaattatga acggtttctt
     5761 ctattttgtc tctatatact acgtatagga aatgtttaca ttttcgtatt gttttcgatt
     5821 cactctatga atagttctta ctacaatttt tttgtctaaa gagtaatact agagataaac
     5881 ataaaaaatg tagaggtcga gtttagatgc aagttcaagg agcgaaaggt ggatgggtag
     5941 gttatatagg gatatagcac agagatatat agcaaagaga tacttttgag caatgtttgt
     6001 ggaagcggta ttcgcaatat tttagtagct cgttacagtc cggtgcgttt ttggtttttt
     6061 gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc tctgaagttc ctatactttc
     6121 tagagaatag gaacttcgga ataggaactt caaagcgttt ccgaaaacga gcgcttccga
     6181 aaatgcaacg cgagctgcgc acatacagct cactgttcac gtcgcaccta tatctgcgtg
     6241 ttgcctgtat atatatatac atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg
     6301 tacttatatg cgtctattta tgtaggatga aaggtagtct agtacctcct gtgatattat
     6361 cccattccat gcggggtatc gtatgcttcc ttcagcacta ccctttagct gttctatatg
     6421 ctgccactcc tcaattggat tagtctcatc cttcaatgct atcatttcct ttgatattgg
     6481 atcatactaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     6541 gccctttcgt c
//
