LOCUS       sequence-335105-        7027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast two-hybrid vector using a copper inducible CUP1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7027)
  AUTHORS   Hernandez J, Ross K, Hamilton B
  TITLE     Inducible Yeast Two-Hybrid with Quantitative Measures
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 7027)
  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 Empty
FEATURES             Location/Qualifiers
     source          1..7027
                     /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"
     terminator      867..914
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      941..1128
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(996..1020)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(1152..1168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1152..1168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1165..1187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1176..1192
                     /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(1200..1206,1207..1224,1225..1230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1245..1266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1383..1400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1554..2142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1634..1653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2471..2518
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2543..2562
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2750..3544)
                     /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(2750..3544)
                     /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(2861..2880)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3471..3490
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3545..3649)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3676..5018
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4639..4686)
                     /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     5059..5077
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5115..5137)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5237..5256
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5277..5558
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             5559..6233
                     /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(6336..6791)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6473..6494)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6685..6704
                     /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 atatggccat ggaggccgaa ttcccgggga
      841 tccgtcgacc tgcagcggcc gcataactag cataacccct tggggcctct aaacgggtct
      901 tgaggggttt tttgcgcgct tgcagccaag ctaattccgg gcgaatttct tatgatttat
      961 gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc
     1021 ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct
     1081 ttctcaggta tagcatgagg tcgctcttat tgaccacacc tctaccggca tgcaagcttg
     1141 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1201 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1261 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1321 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1381 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1441 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1501 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1561 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1621 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1681 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1741 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1801 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1861 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1921 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1981 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2041 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2101 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2161 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2221 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2281 taaagtatat atgagtaacc tgaggctatg gcagggcctg ccgccccgac gttggctgcg
     2341 agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg
     2401 tattggcccc aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc
     2461 ccgcgtttat gaacaaacga cccaacaccg tgcgttttat tctgtctttt tattgccgtc
     2521 atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct ccccctaggg
     2581 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     2641 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     2701 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     2761 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     2821 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     2881 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     2941 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgagtcacg acgagatcct
     3001 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     3061 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     3121 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     3181 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     3241 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     3301 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     3361 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     3421 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     3481 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     3541 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3601 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3661 gaaaagtgcc acctgaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg
     3721 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     3781 gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca
     3841 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat
     3901 gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa
     3961 atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt
     4021 gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga
     4081 agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc
     4141 gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt
     4201 atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga
     4261 ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta
     4321 taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca
     4381 atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta
     4441 gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga
     4501 tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag
     4561 tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt
     4621 ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact tcggaatagg
     4681 aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata
     4741 cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag
     4801 aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag
     4861 gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg
     4921 cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc
     4981 tcatccttca atgctatcat ttcctttgat attggatcat actaagaaac cattattatc
     5041 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     5101 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     5161 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     5221 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tagatcaacg
     5281 acattactat atatataata taggaagcat ttaatagaac agcatcgtaa tatatgtgta
     5341 ctttgcagtt atgacgccag atggcagtag tggaagatat tctttattga aaaatagctt
     5401 gtcaccttac gtacaatctt gatccggagc ttttcttttt ttgccgatta agaattaatt
     5461 cggtcgaaaa aagaaaagga gagggccaag agggagggca ttggtgacta ttgagcacgt
     5521 gagtatacgt gattaagcac acaaaggcag cttggagtat gtctgttatt aatttcacag
     5581 gtagttctgg tccattggtg aaagtttgcg gcttgcagag cacagaggcc gcagaatgtg
     5641 ctctagattc cgatgctgac ttgctgggta ttatatgtgt gcccaataga aagagaacaa
     5701 ttgacccggt tattgcaagg aaaatttcaa gtcttgtaaa agcatataaa aatagttcag
     5761 gcactccgaa atacttggtt ggcgtgtttc gtaatcaacc taaggaggat gttttggctc
     5821 tggtcaatga ttacggcatt gatatcgtcc aactgcatgg agatgagtcg tggcaagaat
     5881 accaagagtt cctcggtttg ccagttatta aaagactcgt atttccaaaa gactgcaaca
     5941 tactactcag tgcagcttca cagaaacctc attcgtttat tcccttgttt gattcagaag
     6001 caggtgggac aggtgaactt ttggattgga actcgatttc tgactgggtt ggaaggcaag
     6061 agagccccga aagcttacat tttatgttag ctggtggact gacgccagaa aatgttggtg
     6121 atgcgcttag attaaatggc gttattggtg ttgatgtaag cggaggtgtg gagacaaatg
     6181 gtgtaaaaga ctctaacaaa atagcaaatt tcgtcaaaaa tgctaagaaa taggttatta
     6241 ctgagtagta tttatttaag tattgtttgt gcacttgccg atctatgcgg tgtgaaatac
     6301 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     6361 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6421 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6481 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6541 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6601 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6661 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6721 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6781 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     6841 gcctcttcgc tattacgcca gatcccatta ccgacatttg ggcgctatac gtgcataagt
     6901 tcatgtatgt atctgtattt aaaacacttt tgtattattt ttcctcatat atgtgtatag
     6961 gtttatacgg atgatttaat tattacttca ccacccttta tttcaggctg atatcttagc
     7021 cttgtta
//
