LOCUS       sequence-442655-        7030 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of two genes in yeast with FLAG and MYC tags and THR1 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7030)
  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 7030)
  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-THR
FEATURES             Location/Qualifiers
     source          1..7030
                     /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"
     rep_origin      complement(1750..2205)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1887..1908)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2099..2118
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2284..2449)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2370..2394
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2597..2620)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2667..3331
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2883..3000
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3295..3318
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3342..3361
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3342..3360
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3378..3407
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      3437..3626
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3698..3715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3869..4457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3949..3968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4628..5419,5420..5488))
                     /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     5251..5270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5489..5593)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5620..6962
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6583..6630)
                     /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     7003..7021
                     /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 caattcccct gcaggcttaa aattatcggt tctagcgatg
      241 ttagagtaca acctctgaac gccgttcctt agcatctttt gttattaatc agctctctgc
      301 tttgcattaa tttgaccacc tgccctgaaa cacctttctt ctagtccacc tgtgtaggct
      361 ttttcacctg aattgttcat tctcgatacg ccctcacgcc ggatgaaaaa tttgtgcaag
      421 agtcatcatc tcgaaaagat atggtgataa aaacaagtgt acttctaacc tgcctaatgg
      481 ttataacagt agcataaagt agatatacgg actacagaaa atggttcgtg ccttcaaaat
      541 taaagttcca gcttcttccg ccaatatcgg ccctggttat gatgttcttg gtgtcggtct
      601 ttctctattc ttggagttag atgtcaccat cgactccagc caagctcagg aaacaaacga
      661 cgatcccaac aactgcaagc tgtcttacac taaagaaagt gaaggctatt ctacggtccc
      721 attgcgttct gatgctaacc tgattaccag gactgcgtta tatgtgttgc gttgcaacaa
      781 tattagaaac ttcccctccg gaaccaaagt tcacgtctcc aacccaatcc cacttggccg
      841 tggattgggt tcctctggtg cagcagttgt ggcaggtgtt attttaggta acgaagtggc
      901 ccaattgggt ttctctaagc aacgtatgtt ggactactgt ttgatgattg aacgtcatcc
      961 agacaacata accgctgcta tgatgggagg cttttgcggt tcattcctaa gggacttgac
     1021 cccacaagag gtggaaagac gtgagattcc attggctgag gtgcttccag aaccttctgg
     1081 tggtgaagat accggtctgg ttcccccatt acctcccacc gatatcggta gacatgtcaa
     1141 ataccaatgg aaccccgcca ttaaatgtat tgcgatcatc ccacagttcg agttgtccac
     1201 cgccgactcc agaggcgttc ttccaaaagc ctacccaacc caggacttgg ttttcaatct
     1261 acaaagattg gccgtcttga ccacagcttt gaccatggac ccacctaatg ccgacttaat
     1321 ctaccctgct atgcaagatc gtgtccacca accttataga aagacattga tcccaggtct
     1381 cacggaaatc ttatcatgtg tcaccccatc cacataccct ggcctattgg gtatctgctt
     1441 gtcaggtgca ggcccaacta tcttggcttt ggccactgag aatttcgaag aaatctctca
     1501 agaaattatc aacaggttcg ccaaaaacgg catcaagtgc tcctggaaac tactggagcc
     1561 tgcctacgat ggtgcaagcg tcgaacagca atgaaatgtc ctttacccct agtatactgt
     1621 gttaaatact attgtagacc atatacatac tgtaattgac cgttaacggt tattatgctg
     1681 agtcttcttc cgttacctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     1741 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     1801 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     1861 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     1921 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     1981 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2041 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2101 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2161 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     2221 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgaa
     2281 ttggagcgac ctcatgctat acctgagaaa gcaacctgac ctacaggaaa gagttactca
     2341 agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat acacttattt
     2401 tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct tatttagaag
     2461 tgtcaacaac gtatctacca acgatttgac ccttttccat cttttcgtaa atttctggca
     2521 aggtagacaa gccgacaacc ttgattggag acttgaccaa acctctggcg aagaattgtt
     2581 aattaagagc tcagatctta tcgtcgtcat ccttgtaatc catcgatact agtgcggccg
     2641 ccctttagtg agggttgaat tcgaattttc aaaaattctt actttttttt tggatggacg
     2701 caaagaagtt taataatcat attacatggc attaccacca tatacatatc catatacata
     2761 tccatatcta atcttactta tatgttgtgg aaatgtaaag agccccatta tcttagccta
     2821 aaaaaacctt ctctttggaa ctttcagtaa tacgcttaac tgctcattgc tatattgaag
     2881 tacggattag aagccgccga gcgggtgaca gccctccgaa ggaagactct cctccgtgcg
     2941 tcctcgtctt caccggtcgc gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg
     3001 aacaataaag attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa
     3061 cctggcccca caaaccttca aatgaacgaa tcaaattaac aaccatagga tgataatgcg
     3121 attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat ttttgatcta
     3181 ttaacagata tataaatgca aaaactgcat aaccacttta actaatactt tcaacatttt
     3241 cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata
     3301 cctctatact ttaacgtcaa ggagaaaaaa ccccggatcc gtaatacgac tcactatagg
     3361 gcccgggcgt cgacatggaa cagaagttga tttccgaaga agacctcgag taagcttggt
     3421 accgcggcta gctaagatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct
     3481 aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa
     3541 tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc
     3601 ttgagaaggt tttgggacgc tcgaagatcc agctgcatta atgaatcggc caacgcgcgg
     3661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4921 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5581 taggggttcc gcgcacattt ccccgaaaag tgccacctga acgaagcatc tgtgcttcat
     5641 tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc
     5701 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct
     5761 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga
     5821 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct
     5881 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat
     5941 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg
     6001 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa
     6061 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt
     6121 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg
     6181 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc
     6241 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc
     6301 actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta gagataaaca
     6361 taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg gatgggtagg
     6421 ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc aatgtttgtg
     6481 gaagcggtat tcgcaatatt ttagtagctc gttacagtcc ggtgcgtttt tggttttttg
     6541 aaagtgcgtc ttcagagcgc ttttggtttt caaaagcgct ctgaagttcc tatactttct
     6601 agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa
     6661 aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat atctgcgtgt
     6721 tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt
     6781 acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg tgatattatc
     6841 ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg ttctatatgc
     6901 tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt tgatattgga
     6961 tcatactaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7021 ccctttcgtc
//
