LOCUS       sequence-204211-        8113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast mating-based Split Ubiquitin Assay, positive control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8113)
  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 8113)
  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: pNubWtXgate
FEATURES             Location/Qualifiers
     source          1..8113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..1344
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(965..1012)
                     /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     1385..1403
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1441..1463)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1563..1582
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1603..1884
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1885..2559
                     /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(2662..3117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3011..3030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3243..3265
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3257..3274
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3258..3274
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3297..4001
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     protein_bind    4126..4150
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     gene            4237..5593
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        4237..4580
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4581..5390
                     /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(4648..4667)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5396..5593
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(5647..5671)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5703..5729
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5751..5938
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5806..5830)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5962..5978)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5962..5978)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5975..5997)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5986..6002
                     /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(6010..6016,6017..6034,6035..6040))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6055..6076
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6193..6210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6364..6952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6444..6463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7123..7914,7915..7983))
                     /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     7746..7765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7984..8088)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt
       61 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt
      121 ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta
      181 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg
      241 ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag
      301 cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat
      361 gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg
      421 gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact
      481 agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga
      541 tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca
      601 gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac
      661 attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa
      721 agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag
      781 gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag
      841 atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt
      901 ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg
      961 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcaaagcgtt
     1021 tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca
     1081 cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt
     1141 gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc
     1201 tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact
     1261 accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc
     1321 tatcatttcc tttgatattg gatcatacta agaaaccatt attatcatga cattaaccta
     1381 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     1441 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     1501 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     1561 tgcggcatca gagcagattg tactgagagt gcaccataga tcaacgacat tactatatat
     1621 ataatatagg aagcatttaa tagaacagca tcgtaatata tgtgtacttt gcagttatga
     1681 cgccagatgg cagtagtgga agatattctt tattgaaaaa tagcttgtca ccttacgtac
     1741 aatcttgatc cggagctttt ctttttttgc cgattaagaa ttaattcggt cgaaaaaaga
     1801 aaaggagagg gccaagaggg agggcattgg tgactattga gcacgtgagt atacgtgatt
     1861 aagcacacaa aggcagcttg gagtatgtct gttattaatt tcacaggtag ttctggtcca
     1921 ttggtgaaag tttgcggctt gcagagcaca gaggccgcag aatgtgctct agattccgat
     1981 gctgacttgc tgggtattat atgtgtgccc aatagaaaga gaacaattga cccggttatt
     2041 gcaaggaaaa tttcaagtct tgtaaaagca tataaaaata gttcaggcac tccgaaatac
     2101 ttggttggcg tgtttcgtaa tcaacctaag gaggatgttt tggctctggt caatgattac
     2161 ggcattgata tcgtccaact gcatggagat gagtcgtggc aagaatacca agagttcctc
     2221 ggtttgccag ttattaaaag actcgtattt ccaaaagact gcaacatact actcagtgca
     2281 gcttcacaga aacctcattc gtttattccc ttgtttgatt cagaagcagg tgggacaggt
     2341 gaacttttgg attggaactc gatttctgac tgggttggaa ggcaagagag ccccgaaagc
     2401 ttacatttta tgttagctgg tggactgacg ccagaaaatg ttggtgatgc gcttagatta
     2461 aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga caaatggtgt aaaagactct
     2521 aacaaaatag caaatttcgt caaaaatgct aagaaatagg ttattactga gtagtattta
     2581 tttaagtatt gtttgtgcac ttgccgatct atgcggtgtg aaataccgca cagatgcgta
     2641 aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2701 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2761 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2821 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2881 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2941 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3001 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3061 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca
     3121 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     3181 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     3241 ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctttcgcgag ctcgatatcc
     3301 ttttgttgtt tccgggtgta caatatggac ttcctctttt ctggcaacca aacccataca
     3361 tcgggattcc tataatacct tcgttggtct ccctaacatg taggtggcgg aggggagata
     3421 tacaatagaa cagataccag acaagacata atgggctaaa caagactaca ccaattacac
     3481 tgcctcattg atggtggtac ataacgaact aatactgtag ccctagactt gatagccatc
     3541 atcatatcga agtttcacta ccctttttcc atttgccatc tattgaagta ataataggcg
     3601 catgcaactt cttttctttt tttttctttt ctctctcccc cgttgttgtc tcaccatatc
     3661 cgcaatgaca aaaaaatgat ggaagacact aaaggaaaaa attaacgaca aagacagcac
     3721 caacagatgt cgttgttcca gagctgatga ggggtatctc gaagcacacg aaactttttc
     3781 cttccttcat tcacgcacac tactctctaa tgagcaacgg tatacggcct tccttccagt
     3841 tacttgaatt tgaaataaaa aaaagtttgc tgtcttgcta tcaagtataa atagacctgc
     3901 aattattaat cttttgtttc ctcgtcattg ttctcgttcc ctttcttcct tgtttctttt
     3961 tctgcacaat atttcaagct ataccaagca tacaatcaac tcgagatgca gattttcgtc
     4021 aagactttga ccggtaaaac cataacattg gaagttgaat cttccgatac catcgacaac
     4081 gttaagtcga aaattcaaga caaggaagga atccctggtg ggtcgacaag tttgtacaaa
     4141 aaagcaggct ctccaaccac catgaattct atacgaagtt attaggtcta gagatctgtt
     4201 tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc
     4261 cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag
     4321 cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga
     4381 agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac
     4441 gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca
     4501 ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc
     4561 acatccgaac ataaacaacc atgggtaagg aaaagactca cgtttcgagg ccgcgattaa
     4621 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     4681 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     4741 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     4801 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     4861 tactcaccac tgcgatcccc ggcaaaacag cattccaggt attagaagaa tatcctgatt
     4921 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     4981 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     5041 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     5101 aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc
     5161 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     5221 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     5281 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     5341 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagtactga
     5401 caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt
     5461 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg
     5521 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct
     5581 ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtgtcga aaacgagctc
     5641 ccgggtaccc agctttcttg tacaaagtgg ttggtggtgg cggatccggt ggaggtggat
     5701 catacccata cgacgttcca gattatgctt aacagccaag ctaattccgg gcgaatttct
     5761 tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt
     5821 aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg
     5881 tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc tctaccggca
     5941 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6001 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6061 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6121 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6301 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6361 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6421 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6481 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6541 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6601 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6661 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6721 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6781 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6841 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6901 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6961 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7021 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7081 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7141 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7201 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7261 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7321 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7381 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7441 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7501 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7561 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7621 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7681 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7741 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7801 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7861 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7921 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7981 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8041 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8101 aaaagtgcca cct
//
