LOCUS       sequence-204210-        8190 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 8190)
  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 8190)
  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: pXNgate21-3HA
FEATURES             Location/Qualifiers
     source          1..8190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        40..745
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     protein_bind    755..779
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     gene            866..2222
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        866..1209
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1210..2019
                     /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(1277..1296)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2025..2222
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(2276..2300)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2446..2535
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      2568..2755
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2623..2647)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2782..2798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2782..2798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2795..2817)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2806..2822
                     /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(2830..2836,2837..2854,2855..2860))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2875..2896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3013..3030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3184..3772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3264..3283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3943..4734,4735..4803))
                     /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     4566..4585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4804..4908)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4935..6277
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5898..5945)
                     /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     6318..6336
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6374..6396)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6496..6515
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6536..6817
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6818..7492
                     /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(7595..8050)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7732..7753)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7944..7963
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(8176..8190,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(8190,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgcc aagctttcgc gagctcgata tccttttgtt gtttccgggt
       61 gtacaatatg gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata
      121 ccttcgttgg tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac
      181 cagacaagac ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg
      241 tacataacga actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca
      301 ctaccctttt tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct
      361 tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa
      421 tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt
      481 tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc
      541 acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat
      601 aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg
      661 tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca
      721 agctatacca agcatacaat caactcatat gtgaacaagt ttgtacaaaa aagcaggctc
      781 tccaaccacc atgaattcta tacgaagtta ttaggtctag agatctgttt agcttgcctc
      841 gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct
      901 tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc
      961 ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta
     1021 cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg
     1081 tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt
     1141 ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca
     1201 taaacaacca tgggtaagga aaagactcac gtttcgaggc cgcgattaaa ttccaacatg
     1261 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     1321 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     1381 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     1441 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     1501 gcgatccccg gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     1561 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     1621 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     1681 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     1741 aaagaaatgc ataagctttt gccattctca ccggattcag tcgtcactca tggtgatttc
     1801 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     1861 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     1921 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     1981 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagtactgac aataaaaaga
     2041 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     2101 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     2161 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     2221 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctcc cgggtaccca
     2281 gctttcttgt acaaagtggt tggtggtggc ggatccggtg gaggtggatc aatgcagatt
     2341 ttcgtcaaga ctttgaccgg taaaaccgga acattggaag ttgaatcttc cgataccatc
     2401 gacaacgtta agtcgaaaat tcaagacaag gaaggaatcc ctggttaccc atacgatgtt
     2461 cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac
     2521 gttccagatt acgctgctca gtgctgaggc gcgccacttc taaataagcg aatttcttat
     2581 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     2641 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     2701 ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagca
     2761 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2821 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2881 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2941 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3001 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3061 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4921 aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga
     4981 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     5041 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa
     5101 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     5161 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa
     5221 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg
     5281 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa
     5341 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg
     5401 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta
     5461 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat
     5521 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat
     5581 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa
     5641 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag
     5701 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat
     5761 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt
     5821 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg
     5881 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga
     5941 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac
     6001 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga
     6061 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg
     6121 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc
     6181 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct
     6241 catccttcaa tgctatcatt tcctttgata ttggatcata ctaagaaacc attattatca
     6301 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     6361 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     6421 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     6481 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat agatcaacga
     6541 cattactata tatataatat aggaagcatt taatagaaca gcatcgtaat atatgtgtac
     6601 tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa aaatagcttg
     6661 tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa gaattaattc
     6721 ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg
     6781 agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg
     6841 tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc
     6901 tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa agagaacaat
     6961 tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa atagttcagg
     7021 cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg ttttggctct
     7081 ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt ggcaagaata
     7141 ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat
     7201 actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc
     7261 aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga
     7321 gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga
     7381 tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg
     7441 tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac
     7501 tgagtagtat ttatttaagt attgtttgtg cacttgccga tctatgcggt gtgaaatacc
     7561 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     7621 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     7681 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     7741 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     7801 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     7861 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     7921 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     7981 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     8041 cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     8101 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     8161 taacgccagg gttttcccag tcacgacgtt
//
