LOCUS       sequence-445552-        7162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with FLAG and MYC tags and MET15
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7162)
  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 7162)
  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
FEATURES             Location/Qualifiers
     source          1..7162
                     /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(2729..2752)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2799..3463
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3015..3132
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3427..3450
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3474..3493
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3474..3492
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3510..3539
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      3569..3758
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3830..3847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4001..4589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4081..4100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4760..5551,5552..5620))
                     /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     5383..5402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5621..5725)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5752..7094
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6715..6762)
                     /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     7135..7153
                     /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 gctcagatct tatcgtcgtc atccttgtaa tccatcgata
     2761 ctagtgcggc cgccctttag tgagggttga attcgaattt tcaaaaattc ttactttttt
     2821 tttggatgga cgcaaagaag tttaataatc atattacatg gcattaccac catatacata
     2881 tccatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat
     2941 tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt
     3001 gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact
     3061 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
     3121 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
     3181 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
     3241 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
     3301 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
     3361 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
     3421 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat ccgtaatacg
     3481 actcactata gggcccgggc gtcgacatgg aacagaagtt gatttccgaa gaagacctcg
     3541 agtaagcttg gtaccgcggc tagctaagat ccgctctaac cgaaaaggaa ggagttagac
     3601 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     3661 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     3721 tgaaaacctt gcttgagaag gttttgggac gctcgaagat ccagctgcat taatgaatcg
     3781 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4441 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5041 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5341 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca
     5761 tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     5821 aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga
     5881 agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa
     5941 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc
     6001 aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt
     6061 ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt
     6121 gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt
     6181 tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg
     6241 cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc
     6301 gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg
     6361 aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat
     6421 tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac
     6481 tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg
     6541 tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag atacttttga
     6601 gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt
     6661 tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt
     6721 cctatacttt ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg
     6781 agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct
     6841 atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat
     6901 gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc
     6961 tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc
     7021 tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc
     7081 tttgatattg gatcatacta agaaaccatt attatcatga cattaaccta taaaaatagg
     7141 cgtatcacga ggccctttcg tc
//
