LOCUS       sequence-442657-        7807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with FLAG and MYC tags and ADE2 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7807)
  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 7807)
  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
FEATURES             Location/Qualifiers
     source          1..7807
                     /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(3374..3397)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        3444..4108
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3660..3777
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4072..4095
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     4119..4138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4119..4137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4155..4184
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      4214..4403
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4475..4492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4646..5234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4726..4745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5405..6196,6197..6265))
                     /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     6028..6047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6266..6370)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6397..7739
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7360..7407)
                     /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     7780..7798
                     /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 gatcttatcg tcgtcatcct tgtaatccat cgatactagt gcggccgccc
     3421 tttagtgagg gttgaattcg aattttcaaa aattcttact ttttttttgg atggacgcaa
     3481 agaagtttaa taatcatatt acatggcatt accaccatat acatatccat atacatatcc
     3541 atatctaatc ttacttatat gttgtggaaa tgtaaagagc cccattatct tagcctaaaa
     3601 aaaccttctc tttggaactt tcagtaatac gcttaactgc tcattgctat attgaagtac
     3661 ggattagaag ccgccgagcg ggtgacagcc ctccgaagga agactctcct ccgtgcgtcc
     3721 tcgtcttcac cggtcgcgtt cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac
     3781 aataaagatt ctacaatact agcttttatg gttatgaaga ggaaaaattg gcagtaacct
     3841 ggccccacaa accttcaaat gaacgaatca aattaacaac cataggatga taatgcgatt
     3901 agttttttag ccttatttct ggggtaatta atcagcgaag cgatgatttt tgatctatta
     3961 acagatatat aaatgcaaaa actgcataac cactttaact aatactttca acattttcgg
     4021 tttgtattac ttcttattca aatgtaataa aagtatcaac aaaaaattgt taatatacct
     4081 ctatacttta acgtcaagga gaaaaaaccc cggatccgta atacgactca ctatagggcc
     4141 cgggcgtcga catggaacag aagttgattt ccgaagaaga cctcgagtaa gcttggtacc
     4201 gcggctagct aagatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg
     4261 tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt
     4321 ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg
     4381 agaaggtttt gggacgctcg aagatccagc tgcattaatg aatcggccaa cgcgcgggga
     4441 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4501 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4561 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4621 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4681 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4741 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4801 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4861 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4921 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4981 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5041 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5101 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5161 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5221 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5281 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5341 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5401 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5461 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5521 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5581 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5641 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5701 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5761 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5821 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5881 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5941 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6001 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6061 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6121 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6181 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6241 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6301 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6361 gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt
     6421 gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     6481 tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca
     6541 tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     6601 gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata
     6661 cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt
     6721 agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac
     6781 tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa
     6841 aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc
     6901 aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac
     6961 agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat
     7021 tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact
     7081 ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa
     7141 aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta
     7201 tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa
     7261 gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa
     7321 gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctaga
     7381 gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat
     7441 gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc
     7501 ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact
     7561 tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca
     7621 ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc
     7681 cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga tattggatca
     7741 tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7801 tttcgtc
//
