LOCUS       sequence-445559-        7183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with V5 and HA tags and MET15 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7183)
  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 7183)
  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-MET (V5 and HA)
FEATURES             Location/Qualifiers
     source          1..7183
                     /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"
     CDS             442..1776
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNG
                     FGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
     rep_origin      complement(1882..2337)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2019..2040)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2231..2250
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2416..2581)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2502..2526
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2726..2767)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        2820..3484
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3036..3153
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3448..3471
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3495..3514
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3495..3513
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3537..3563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3590..3779
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3851..3868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4022..4610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4102..4121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4781..5572,5573..5641))
                     /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     5404..5423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5642..5746)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5773..7115
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6736..6783)
                     /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     7156..7174
                     /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 caattcagcc atcctcatga aaactgtgta acataataac
      241 cgaagtgtcg aaaaggtggc accttgtcca attgaacacg ctcgatgaaa aaaataagat
      301 atatataagg ttaagtaaag cgtctgttag aaaggaagtt tttccttttt cttgctctct
      361 tgtcttttca tctactattt ccttcgtgta atacagggtc gtcagataca tagatacaat
      421 tctattaccc ccatccatac aatgccatct catttcgata ctgttcaact acacgccggc
      481 caagagaacc ctggtgacaa tgctcacaga tccagagctg taccaattta cgccaccact
      541 tcttatgttt tcgaaaactc taagcatggt tcgcaattgt ttggtctaga agttccaggt
      601 tacgtctatt cccgtttcca aaacccaacc agtaatgttt tggaagaaag aattgctgct
      661 ttagaaggtg gtgctgctgc tttggctgtt tcctccggtc aagccgctca aacccttgcc
      721 atccaaggtt tggcacacac tggtgacaac atcgtttcca cttcttactt atacggtggt
      781 acttataacc agttcaaaat ctcgttcaaa agatttggta tcgaggctag atttgttgaa
      841 ggtgacaatc cagaagaatt tgaaaaggtc tttgatgaaa gaaccaaggc tgtttatttg
      901 gaaaccattg gtaatccaaa gtacaatgtt ccggattttg aaaaaattgt tgcaattgct
      961 cacaaacacg gtattccagt tgtcgttgac aacacatttg gtgccggtgg ttacttctgt
     1021 cagccaatta aatacggtgc tgatattgta acacattctg ctaccaaatg gattggtggt
     1081 catggtacta ctatcggtgg tattattgtt gactctggta agttcccatg gaaggactac
     1141 ccagaaaagt tccctcaatt ctctcaacct gccgaaggat atcacggtac tatctacaat
     1201 gaagcctacg gtaacttggc atacatcgtt catgttagaa ctgaactatt aagagatttg
     1261 ggtccattga tgaacccatt tgcctctttc ttgctactac aaggtgttga aacattatct
     1321 ttgagagctg aaagacacgg tgaaaatgca ttgaagttag ccaaatggtt agaacaatcc
     1381 ccatacgtat cttgggtttc ataccctggt ttagcatctc attctcatca tgaaaatgct
     1441 aagaagtatc tatctaacgg tttcggtggt gtcttatctt tcggtgtaaa agacttacca
     1501 aatgccgaca aggaaactga cccattcaaa ctttctggtg ctcaagttgt tgacaattta
     1561 aagcttgcct ctaacttggc caatgttggt gatgccaaga ccttagtcat tgctccatac
     1621 ttcactaccc acaaacaatt aaatgacaaa gaaaagttgg catctggtgt taccaaggac
     1681 ttaattcgtg tctctgttgg tatcgaattt attgatgaca ttattgcaga cttccagcaa
     1741 tcttttgaaa ctgttttcgc tggccaaaaa ccatgagtgt gcgtaatgag ttgtaaaatt
     1801 atgtataaac ctactttctc tcacaagtat atgcggtgtg aaataccgca cagatgcgta
     1861 aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1921 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1981 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2041 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2101 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2161 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2221 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2281 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca
     2341 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     2401 acgccagctg aattggagcg acctcatgct atacctgaga aagcaacctg acctacagga
     2461 aagagttact caagaataag aattttcgtt ttaaaaccta agagtcactt taaaatttgt
     2521 atacacttat tttttttata acttatttaa taataaaaat cataaatcat aagaaattcg
     2581 cttatttaga agtgtcaaca acgtatctac caacgatttg acccttttcc atcttttcgt
     2641 aaatttctgg caaggtagac aagccgacaa ccttgattgg agacttgacc aaacctctgg
     2701 cgaagaattg ttaattaaga gctcaggtag aatccagacc cagcagaggg ttagggatag
     2761 gcttaccaga tctcatcgat actagtgcgg ccgcccttta gtgagggttg aattcgaatt
     2821 ttcaaaaatt cttacttttt ttttggatgg acgcaaagaa gtttaataat catattacat
     2881 ggcattacca ccatatacat atccatatac atatccatat ctaatcttac ttatatgttg
     2941 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag
     3001 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggtg
     3061 acagccctcc gaaggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg
     3121 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct
     3181 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatgaac
     3241 gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg
     3301 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaaactg
     3361 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg
     3421 taataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa
     3481 aaaccccgga tccgtaatac gactcactat agggcccggg cgtcgacatg ctcgagtacc
     3541 catacgatgt tccagattac gcttaagctt ggtaccgcgg ctagctaaga tccgctctaa
     3601 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     3661 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     3721 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga
     3781 tccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4021 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4081 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4201 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4441 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4621 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4681 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4741 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4801 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4861 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4921 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4981 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5041 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5101 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5161 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5221 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5281 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5341 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5401 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5461 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5521 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5581 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5641 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5701 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5761 aagtgccacc tgaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga
     5821 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     5881 aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg
     5941 cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca
     6001 acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg
     6061 catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg
     6121 cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga
     6181 aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt
     6241 tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata
     6301 ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc
     6361 ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag
     6421 gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt
     6481 tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat
     6541 gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat
     6601 atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag
     6661 ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt
     6721 tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     6781 ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc gcacatacag
     6841 ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat acatgagaag
     6901 aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt tatgtaggat
     6961 gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta tcgtatgctt
     7021 ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg attagtctca
     7081 tccttcaatg ctatcatttc ctttgatatt ggatcatact aagaaaccat tattatcatg
     7141 acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
