LOCUS       sequence-335101-        7713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast two-hybrid vector using a copper inducible CUP1 promoter, 
            expressing mGrape3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7713)
  AUTHORS   Hernandez J, Ross K, Hamilton B
  TITLE     Inducible Yeast Two-Hybrid with Quantitative Measures
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 7713)
  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: "bioRxiv"
COMMENT     Sequence Label: pGBK CUP1 mGrape3
FEATURES             Location/Qualifiers
     source          1..7713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..235
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             291..731
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     primer_bind     742..761
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        742..760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             780..809
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(958..976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1226..1245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1420..1443
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      1553..1600
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1627..1814
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1682..1706)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(1838..1854)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1838..1854)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1851..1873)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1862..1878
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1886..1892,1893..1910,1911..1916))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1931..1952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2069..2086)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2240..2828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2320..2339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3157..3204
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3229..3248
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3436..4230)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3436..4230)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3547..3566)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4157..4176
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4231..4335)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4362..5704
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5325..5372)
                     /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     5745..5763
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5801..5823)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5923..5942
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5963..6244
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6245..6919
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(7022..7477)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7159..7180)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7371..7390
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctagttagaa aaagacattt ttgctgtcag tcactgtcaa gagattcttt tgctggcatt
       61 tcttctagaa gcaaaaagag cgatgcgtct tttccgctga accgttccag caaaaaagac
      121 taccaacgca atatggattg tcagaatcat ataaaagaga agcaaataac tccttgtctt
      181 gtatcaattg cattataata tcttcttgtt agtgcaatat catatagaag tcatcgaaat
      241 agatattaag aaaaacaaac tgtccaagct tgaagcaagc ctcctgaaag atgaagctac
      301 tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag tgctccaaag
      361 aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac tctcccaaaa
      421 ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg ctagaaagac
      481 tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt ttgaaaatgg
      541 attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat aatgtgaata
      601 aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta acattgagac
      661 agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt caaagacagt
      721 tgactgtatc gccggaattt gtaatacgac tcactatagg gcgagccgcc atcatggagg
      781 agcagaagct gatctcagag gaggacctgc atatggtgag caagggcgag gagaataaca
      841 tggccgtcat caaggagttc atgcgcttca agacgcacat ggagggctcc gtgaacggcc
      901 acgagttcga gatcgagggc aagggcgagg gccgccccta cgagggcacc cagaccgcca
      961 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcaga
     1021 tgatgtacgg ctccaaggcc tacgtgaagc acccccctga catccccgac tacatgaagc
     1081 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1141 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1201 acggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1261 cctcctccga gcggttgtac cccgaggacg gcgccctgaa gggcgaggtc aagatgaggc
     1321 tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga
     1381 agcccgtgca gctgcccggc gcctacaagc tcgactacaa gctggacatc acctcccaca
     1441 acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg
     1501 gcatggacga gctgtacaag taaggatccg tcgacctgca gcggccgcat aactagcata
     1561 accccttggg gcctctaaac gggtcttgag gggttttttg cgcgcttgca gccaagctaa
     1621 ttccgggcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     1681 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     1741 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac
     1801 cacacctcta ccggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1861 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1921 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1981 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2041 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2101 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2161 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2221 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2281 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2341 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2401 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2461 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2521 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2581 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2641 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2701 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2761 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2821 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2881 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2941 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaacctgag gctatggcag
     3001 ggcctgccgc cccgacgttg gctgcgagcc ctgggccttc acccgaactt ggggggtggg
     3061 gtggggaaaa ggaagaaacg cgggcgtatt ggccccaatg gggtctcggt ggggtatcga
     3121 cagagtgcca gccctgggac cgaaccccgc gtttatgaac aaacgaccca acaccgtgcg
     3181 ttttattctg tctttttatt gccgtcatag cgcgggttcc ttccggtatt gtctccttcc
     3241 gtgtttcagt tagcctcccc ctagggtggg cgaagaactc cagcatgaga tccccgcgct
     3301 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg
     3361 cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga
     3421 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     3481 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     3541 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     3601 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     3661 atcgccatga gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa
     3721 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     3781 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     3841 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     3901 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     3961 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     4021 cagccacgat agccgcgctg cctcgtcttg cagttcattc agggcaccgg acaggtcggt
     4081 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     4141 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     4201 acctgcgtgc aatccatctt gttcaatcat actcttcctt tttcaatatt attgaagcat
     4261 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4321 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc
     4381 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     4441 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg
     4501 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     4561 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat
     4621 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc
     4681 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt
     4741 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat
     4801 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt
     4861 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg
     4921 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct
     4981 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat
     5041 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa
     5101 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta
     5161 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg
     5221 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt
     5281 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt
     5341 ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg
     5401 aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt
     5461 gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc
     5521 gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta
     5581 tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat
     5641 gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg
     5701 gatcatacta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5761 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     5821 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     5881 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     5941 tactgagagt gcaccataga tcaacgacat tactatatat ataatatagg aagcatttaa
     6001 tagaacagca tcgtaatata tgtgtacttt gcagttatga cgccagatgg cagtagtgga
     6061 agatattctt tattgaaaaa tagcttgtca ccttacgtac aatcttgatc cggagctttt
     6121 ctttttttgc cgattaagaa ttaattcggt cgaaaaaaga aaaggagagg gccaagaggg
     6181 agggcattgg tgactattga gcacgtgagt atacgtgatt aagcacacaa aggcagcttg
     6241 gagtatgtct gttattaatt tcacaggtag ttctggtcca ttggtgaaag tttgcggctt
     6301 gcagagcaca gaggccgcag aatgtgctct agattccgat gctgacttgc tgggtattat
     6361 atgtgtgccc aatagaaaga gaacaattga cccggttatt gcaaggaaaa tttcaagtct
     6421 tgtaaaagca tataaaaata gttcaggcac tccgaaatac ttggttggcg tgtttcgtaa
     6481 tcaacctaag gaggatgttt tggctctggt caatgattac ggcattgata tcgtccaact
     6541 gcatggagat gagtcgtggc aagaatacca agagttcctc ggtttgccag ttattaaaag
     6601 actcgtattt ccaaaagact gcaacatact actcagtgca gcttcacaga aacctcattc
     6661 gtttattccc ttgtttgatt cagaagcagg tgggacaggt gaacttttgg attggaactc
     6721 gatttctgac tgggttggaa ggcaagagag ccccgaaagc ttacatttta tgttagctgg
     6781 tggactgacg ccagaaaatg ttggtgatgc gcttagatta aatggcgtta ttggtgttga
     6841 tgtaagcgga ggtgtggaga caaatggtgt aaaagactct aacaaaatag caaatttcgt
     6901 caaaaatgct aagaaatagg ttattactga gtagtattta tttaagtatt gtttgtgcac
     6961 ttgccgatct atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     7021 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     7081 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     7141 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     7201 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     7261 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     7321 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     7381 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     7441 acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
     7501 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagatc ccattaccga
     7561 catttgggcg ctatacgtgc ataagttcat gtatgtatct gtatttaaaa cacttttgta
     7621 ttatttttcc tcatatatgt gtataggttt atacggatga tttaattatt acttcaccac
     7681 cctttatttc aggctgatat cttagccttg tta
//
