LOCUS       sequence-203463-        8172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast mating-based Split Ubiquitin Assay, prey vector for in vivo 
            cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8172)
  AUTHORS   Obrdlik P, El-Bakkoury M, Hamacher T, Cappellaro C, Vilarino C, 
            Fleischer C, Ellerbrok H, Kamuzinzi R, Ledent V, Blaudez D, Sanders 
            D, Revuelta JL, Boles E, Andre B, Frommer WB
  TITLE     K+ channel interactions detected by a genetic system optimized for 
            systematic studies of membrane protein interactions.
  JOURNAL   Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12242-7. Epub 2004 Aug
            6.
  PUBMED    15299147
REFERENCE   2  (bases 1 to 8172)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2004-08-17"; volume: "101(33)"; pages: 
            "12242-7. Epub 2004 Aug 6"
COMMENT     Sequence Label: pNXgate32-3HA
FEATURES             Location/Qualifiers
     source          1..8172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        180..884
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     protein_bind    1009..1033
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     gene            1120..2476
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1120..1463
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1464..2273
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1531..1550)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2279..2476
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(2530..2554)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2568..2657
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      2690..2877
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2745..2769)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2904..2920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2904..2920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2917..2939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2928..2944
                     /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(2952..2958,2959..2976,2977..2982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2997..3018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3135..3152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3306..3894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3386..3405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4065..4856,4857..4925))
                     /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     4688..4707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4926..5030)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5057..6399
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6020..6067)
                     /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     6440..6458
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6496..6518)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6618..6637
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6658..6939
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6940..7614
                     /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(7717..8172)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7854..7875)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8066..8085
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctttcgc gagctcgata
      181 tccttttgtt gtttccgggt gtacaatatg gacttcctct tttctggcaa ccaaacccat
      241 acatcgggat tcctataata ccttcgttgg tctccctaac atgtaggtgg cggaggggag
      301 atatacaata gaacagatac cagacaagac ataatgggct aaacaagact acaccaatta
      361 cactgcctca ttgatggtgg tacataacga actaatactg tagccctaga cttgatagcc
      421 atcatcatat cgaagtttca ctaccctttt tccatttgcc atctattgaa gtaataatag
      481 gcgcatgcaa cttcttttct ttttttttct tttctctctc ccccgttgtt gtctcaccat
      541 atccgcaatg acaaaaaaat gatggaagac actaaaggaa aaaattaacg acaaagacag
      601 caccaacaga tgtcgttgtt ccagagctga tgaggggtat ctcgaagcac acgaaacttt
      661 ttccttcctt cattcacgca cactactctc taatgagcaa cggtatacgg ccttccttcc
      721 agttacttga atttgaaata aaaaaaagtt tgctgtcttg ctatcaagta taaatagacc
      781 tgcaattatt aatcttttgt ttcctcgtca ttgttctcgt tccctttctt ccttgtttct
      841 ttttctgcac aatatttcaa gctataccaa gcatacaatc aactcgagat gcagattttc
      901 gtcaagactt tgaccggtaa aaccggaaca ttggaagttg aatcttccga taccatcgac
      961 aacgttaagt cgaaaattca agacaaggaa ggaatccctg gtgggtcgac aagtttgtac
     1021 aaaaaagcag gctctccaac caccatgaat tctatacgaa gttattaggt ctagagatct
     1081 gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc
     1141 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
     1201 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
     1261 cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca
     1321 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
     1381 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
     1441 atcacatccg aacataaaca accatgggta aggaaaagac tcacgtttcg aggccgcgat
     1501 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     1561 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     1621 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1681 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     1741 ggttactcac cactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
     1801 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     1861 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     1921 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     1981 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
     2041 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2101 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2161 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2221 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagtac
     2281 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     2341 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     2401 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     2461 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt cgaaaacgag
     2521 ctcccgggta cccagctttc ttgtacaaag tggttggtgg tggcggatac ccatacgatg
     2581 ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg
     2641 acgttccaga ttacgctgct cagtgctgag gcgcgccact tctaaataag cgaatttctt
     2701 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     2761 aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt
     2821 caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct ctaccggcag
     2881 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2941 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3001 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3061 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3121 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3181 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3241 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3301 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3361 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3421 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3481 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3541 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3601 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3661 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3721 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3781 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3841 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3901 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3961 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4021 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4081 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4141 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4201 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4261 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4321 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4381 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4441 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4501 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4561 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4621 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4681 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4741 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4801 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4861 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4921 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4981 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5041 cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc
     5101 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     5161 cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc
     5221 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     5281 tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa
     5341 aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt
     5401 tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag
     5461 aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg
     5521 cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat
     5581 tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg
     5641 attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt
     5701 ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac
     5761 aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt
     5821 agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag
     5881 atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta
     5941 gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt
     6001 tggttttcaa aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag
     6061 gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat
     6121 acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga
     6181 gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta
     6241 ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat
     6301 gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt
     6361 ctcatccttc aatgctatca tttcctttga tattggatca tactaagaaa ccattattat
     6421 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     6481 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6541 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6601 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atagatcaac
     6661 gacattacta tatatataat ataggaagca tttaatagaa cagcatcgta atatatgtgt
     6721 actttgcagt tatgacgcca gatggcagta gtggaagata ttctttattg aaaaatagct
     6781 tgtcacctta cgtacaatct tgatccggag cttttctttt tttgccgatt aagaattaat
     6841 tcggtcgaaa aaagaaaagg agagggccaa gagggagggc attggtgact attgagcacg
     6901 tgagtatacg tgattaagca cacaaaggca gcttggagta tgtctgttat taatttcaca
     6961 ggtagttctg gtccattggt gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt
     7021 gctctagatt ccgatgctga cttgctgggt attatatgtg tgcccaatag aaagagaaca
     7081 attgacccgg ttattgcaag gaaaatttca agtcttgtaa aagcatataa aaatagttca
     7141 ggcactccga aatacttggt tggcgtgttt cgtaatcaac ctaaggagga tgttttggct
     7201 ctggtcaatg attacggcat tgatatcgtc caactgcatg gagatgagtc gtggcaagaa
     7261 taccaagagt tcctcggttt gccagttatt aaaagactcg tatttccaaa agactgcaac
     7321 atactactca gtgcagcttc acagaaacct cattcgttta ttcccttgtt tgattcagaa
     7381 gcaggtggga caggtgaact tttggattgg aactcgattt ctgactgggt tggaaggcaa
     7441 gagagccccg aaagcttaca ttttatgtta gctggtggac tgacgccaga aaatgttggt
     7501 gatgcgctta gattaaatgg cgttattggt gttgatgtaa gcggaggtgt ggagacaaat
     7561 ggtgtaaaag actctaacaa aatagcaaat ttcgtcaaaa atgctaagaa ataggttatt
     7621 actgagtagt atttatttaa gtattgtttg tgcacttgcc gatctatgcg gtgtgaaata
     7681 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt
     7741 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     7801 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     7861 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     7921 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     7981 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     8041 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     8101 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     8161 tacagggcgc gt
//
