LOCUS       sequence-204212-        8115 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 8115)
  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 8115)
  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: pXNubWtgate
FEATURES             Location/Qualifiers
     source          1..8115
                     /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    4011..4035
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     gene            4122..5478
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        4122..4465
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4466..5275
                     /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(4533..4552)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5281..5478
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(5532..5556)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5702..5728
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5753..5940
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5808..5832)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5964..5980)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5964..5980)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5977..5999)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5988..6004
                     /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(6012..6018,6019..6036,6037..6042))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6057..6078
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6195..6212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6366..6954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6446..6465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7125..7916,7917..7985))
                     /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     7748..7767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7986..8090)
                     /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 tcatatgtga acaagtttgt
     4021 acaaaaaagc aggctctcca accaccatga attctatacg aagttattag gtctagagat
     4081 ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata
     4141 ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca
     4201 tttagcccat acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg
     4261 cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca
     4321 cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt
     4381 tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc
     4441 acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt cgaggccgcg
     4501 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     4561 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     4621 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     4681 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     4741 atggttactc accactgcga tccccggcaa aacagcattc caggtattag aagaatatcc
     4801 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     4861 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     4921 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     4981 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
     5041 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     5101 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     5161 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     5221 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcagt
     5281 actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt
     5341 gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc
     5401 atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga
     5461 atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgaaaacg
     5521 agctcccggg tacccagctt tcttgtacaa agtggttggt ggtggcggat ccggtggagg
     5581 tggatcaatg cagattttcg tcaagacttt gaccggtaaa accataacat tggaagttga
     5641 atcttccgat accatcgaca acgttaagtc gaaaattcaa gacaaggaag gaatccctgg
     5701 ttacccatac gacgttccag attatgctta gctgcagcca agctaattcc gggcgaattt
     5761 cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt
     5821 ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta
     5881 ggtcaggttg ctttctcagg tatagcatga ggtcgctctt attgaccaca cctctaccgg
     5941 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     6001 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     6061 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6121 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6181 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6241 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6301 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6361 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6421 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6481 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6541 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6601 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6661 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6721 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6781 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6841 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6901 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6961 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7021 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7081 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7141 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7201 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7261 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7321 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7381 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7441 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7501 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7561 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7621 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7681 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7741 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7801 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7861 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7921 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7981 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     8041 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8101 cgaaaagtgc cacct
//
