LOCUS       sequence-445558-        7828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and ADE2 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7828)
  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 7828)
  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-ADE (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..7828
                     /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        208..583
                     /gene="S. cerevisiae ADE2"
                     /label=ADE2 promoter
     CDS             584..2299
                     /codon_start=1
                     /gene="S. cerevisiae ADE2"
                     /product="phosphoribosylaminoimidazole carboxylase, 
                     required for adenine biosynthesis"
                     /label=ADE2
                     /note="yeast auxotrophic marker"
                     /translation="MDSRTVGILGGGQLGRMIVEAANRLNIKTVILDAENSPAKQISNS
                     NDHVNGSFSNPLDIEKLAEKCDVLTIEIEHVDVPTLKNLQVKHPKLKIYPSPETIRLIQ
                     DKYIQKEHLIKNGIAVTQSVPVEQASETSLLNVGRDLGFPFVLKSRTLAYDGRGNFVVK
                     NKEMIPEALEVLKDRPLYAEKWAPFTKELAVMIVRSVNGLVFSYPIVETIHKDNICDLC
                     YASARVPDSVQLKAKLLAENAIKSFPGCGIFGVEMFYLETGELLINEIAPRPHNSGHYT
                     IDACVTSQFEAHLRSILDLPMPKNFTSFSTITTNAIMLNVLGDKHTKDKELETCERALA
                     TPGSSVYLYGKESRPNRKVGHINIIASSMAECEQRLNYITGRTDIPIKISVAQKLDLEA
                     MVKPLVGIIMGSDSDLPVMSAACAVLKDFGVPFEVTIVSAHRTPHRMSAYAISASKRGI
                     KTIIAGAGGAAHLPGMVAAMTPLPVIGVPVKGSCLDGVDSLHSIVQMPRGVPVATVAIN
                     NSTNAALLAVRLLGAYDSSYTTKMEQFLLKQEEEVLVKAQKLETVGYEAYLENK"
     rep_origin      complement(2527..2982)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2664..2685)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2876..2895
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3061..3226)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3147..3171
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3371..3412)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        3465..4129
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3681..3798
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4093..4116
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     4140..4159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4140..4158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4182..4208
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4235..4424
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4496..4513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4667..5255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4747..4766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5426..6217,6218..6286))
                     /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     6049..6068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6287..6391)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6418..7760
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7381..7428)
                     /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     7801..7819
                     /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 caattcattc ttgaataata cataactttt cttaaaagaa
      241 tcaaagacag ataaaattta agagatatta aatattagtg agaagccgag aattttgtaa
      301 caccaacata acactgacat ctttaacaac ttttaattat gatacatttc ttacgtcatg
      361 attgattatt acagctatgc tgacaaatga ctcttgttgc atggctacga accgggtaat
      421 actaagtgat tgactcttgc tgacctttta ttaagaacta aatggacaat attatggagc
      481 atttcatgta taaattggtg cgtaaaatcg ttggatctct cttctaagta catcctacta
      541 taacaatcaa gaaaaacaag aaaatcggac aaaacaatca agtatggatt ctagaacagt
      601 tggtatatta ggagggggac aattgggacg tatgattgtt gaggcagcaa acaggctcaa
      661 cattaagacg gtaatactag atgctgaaaa ttctcctgcc aaacaaataa gcaactccaa
      721 tgaccacgtt aatggctcct tttccaatcc tcttgatatc gaaaaactag ctgaaaaatg
      781 tgatgtgcta acgattgaga ttgagcatgt tgatgttcct acactaaaga atcttcaagt
      841 aaaacatccc aaattaaaaa tttacccttc tccagaaaca atcagattga tacaagacaa
      901 atatattcaa aaagagcatt taatcaaaaa tggtatagca gttacccaaa gtgttcctgt
      961 ggaacaagcc agtgagacgt ccctattgaa tgttggaaga gatttgggtt ttccattcgt
     1021 cttgaagtcg aggactttgg catacgatgg aagaggtaac ttcgttgtaa agaataagga
     1081 aatgattccg gaagctttgg aagtactgaa ggatcgtcct ttgtacgccg aaaaatgggc
     1141 accatttact aaagaattag cagtcatgat tgtgaggtct gttaacggtt tagtgttttc
     1201 ttacccaatt gtagagacta tccacaagga caatatttgt gacttatgtt atgcgtctgc
     1261 tagagttccg gactccgttc aacttaaggc gaagttgttg gcagaaaatg caatcaaatc
     1321 ttttcccggt tgtggtatat ttggtgtgga aatgttctat ttagaaacag gggaattgct
     1381 tattaacgaa attgccccaa ggcctcacaa ctctggacat tataccattg atgcttgcgt
     1441 cacttctcaa tttgaagctc atttgagatc aatattggat ttgccaatgc caaagaattt
     1501 cacatctttc tccaccatta caacgaacgc cattatgcta aatgttcttg gagacaaaca
     1561 tacaaaagat aaagagctag aaacttgcga aagagcattg gcgactccag gttcctcagt
     1621 gtacttatat ggaaaagagt ctagacctaa cagaaaagta ggtcacataa atattattgc
     1681 ctccagtatg gcggaatgtg aacaaaggct gaactacatt acaggtagaa ctgatattcc
     1741 aatcaaaatc tctgtcgctc aaaagttgga cttggaagca atggtcaaac cattggttgg
     1801 aatcatcatg ggatcagact ctgacttgcc ggtaatgtct gccgcatgtg cggttttaaa
     1861 agattttggc gttccatttg aagtgacaat agtctctgct catagaactc cacataggat
     1921 gtcagcatat gctatttccg caagcaagcg tggaattaaa acaattatcg ctggagctgg
     1981 tggggctgct cacttgccag gtatggtggc tgcaatgaca ccacttcctg tcatcggtgt
     2041 gcccgtaaaa ggttcttgtc tagatggagt agattcttta cattcaattg tgcaaatgcc
     2101 tagaggtgtt ccagtagcta ccgtcgctat taataatagt acgaacgctg cgctgttggc
     2161 tgtcagactg cttggcgctt atgattcaag ttatacaacg aaaatggaac agtttttatt
     2221 aaagcaagaa gaagaagttc ttgtcaaagc acaaaagtta gaaactgtcg gttacgaagc
     2281 ttatctagaa aacaagtaat atataagttt attgatatac ttgtacagca aataattata
     2341 aaatgatata cctatttttt aggctttgtt atgattacat caaatgtgga cttcatacat
     2401 agaaatcaac gcttacaggt gtcctttttt aagaatttca tacataagat caatgcggta
     2461 tttcacaccg catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     2521 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2581 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2641 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2701 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2761 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2821 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2881 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2941 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     3001 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgaattg
     3061 gagcgacctc atgctatacc tgagaaagca acctgaccta caggaaagag ttactcaaga
     3121 ataagaattt tcgttttaaa acctaagagt cactttaaaa tttgtataca cttatttttt
     3181 ttataactta tttaataata aaaatcataa atcataagaa attcgcttat ttagaagtgt
     3241 caacaacgta tctaccaacg atttgaccct tttccatctt ttcgtaaatt tctggcaagg
     3301 tagacaagcc gacaaccttg attggagact tgaccaaacc tctggcgaag aattgttaat
     3361 taagagctca ggtagaatcc agacccagca gagggttagg gataggctta ccagatctca
     3421 tcgatactag tgcggccgcc ctttagtgag ggttgaattc gaattttcaa aaattcttac
     3481 tttttttttg gatggacgca aagaagttta ataatcatat tacatggcat taccaccata
     3541 tacatatcca tatacatatc catatctaat cttacttata tgttgtggaa atgtaaagag
     3601 ccccattatc ttagcctaaa aaaaccttct ctttggaact ttcagtaata cgcttaactg
     3661 ctcattgcta tattgaagta cggattagaa gccgccgagc gggtgacagc cctccgaagg
     3721 aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc
     3781 tcgcgccgca ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag
     3841 aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa tgaacgaatc aaattaacaa
     3901 ccataggatg ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa
     3961 gcgatgattt ttgatctatt aacagatata taaatgcaaa aactgcataa ccactttaac
     4021 taatactttc aacattttcg gtttgtatta cttcttattc aaatgtaata aaagtatcaa
     4081 caaaaaattg ttaatatacc tctatacttt aacgtcaagg agaaaaaacc ccggatccgt
     4141 aatacgactc actatagggc ccgggcgtcg acatgctcga gtacccatac gatgttccag
     4201 attacgctta agcttggtac cgcggctagc taagatccgc tctaaccgaa aaggaaggag
     4261 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     4321 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     4381 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaagatccag ctgcattaat
     4441 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4501 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4561 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4621 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4681 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4741 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4801 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4861 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4921 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4981 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5041 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5101 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5161 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5221 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5281 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5341 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5401 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5461 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5521 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5581 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5641 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5701 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5761 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5821 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5881 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5941 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6001 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6061 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6121 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6181 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6241 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6301 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6361 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac
     6421 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     6481 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     6541 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     6601 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     6661 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     6721 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     6781 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     6841 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     6901 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     6961 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     7021 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     7081 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     7141 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     7201 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     7261 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     7321 tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct
     7381 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg
     7441 aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc
     7501 gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt
     7561 gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt
     7621 acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc
     7681 tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc
     7741 atttcctttg atattggatc atactaagaa accattatta tcatgacatt aacctataaa
     7801 aataggcgta tcacgaggcc ctttcgtc
//
