LOCUS       sequence-203462-        6681 bp    DNA     circular SYN 15-APR-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 6681)
  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 6681)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pNXgate33-3HA
FEATURES             Location/Qualifiers
     source          1..6681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    288..309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(324..329,330..347,348..354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    362..378
                     /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)."
     primer_bind     367..389
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     386..402
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     386..402
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    908..932
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     gene            1019..2375
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1019..1362
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1363..2172
                     /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(1430..1449)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2178..2375
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(2429..2453)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2467..2556
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     misc_feature    3074..4236
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN4 centromere fused to the 
                     autonomously replicating sequence ARS1/ARS416"
     rep_origin      3399..4236
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1/ARS416"
     CDS             complement(4075..4749)
                     /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"
     promoter        complement(4750..4851)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4871..4889)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4957..5061
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5062..5130,5131..5922)
                     /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     complement(5280..5299)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6093..6681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6582..6601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      241 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      301 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      361 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
      421 gcatgcgttg tctcaccata tccgcaatga caaaaaaatg atggaagaca ctaaaggaaa
      481 aaattaacga caaagacagc accaacagat gtcgttgttc cagagctgat gaggggtatc
      541 tcgaagcaca cgaaactttt tccttccttc attcacgcac actactctct aatgagcaac
      601 ggtatacggc cttccttcca gttacttgaa tttgaaataa aaaaaagttt gctgtcttgc
      661 tatcaagtat aaatagacct gcaattatta atcttttgtt tcctcgtcat tgttctcgtt
      721 ccctttcttc cttgtttctt tttctgcaca atatttcaag ctataccaag catacaatca
      781 actcgagatg cagattttcg tcaagacttt gaccggtaaa accggaacat tggaagttga
      841 atcttccgat accatcgaca acgttaagtc gaaaattcaa gacaaggaag gaatccctgg
      901 tgggtcgaca agtttgtaca aaaaagcagg ctctccaacc accatgaatt ctatacgaag
      961 ttattaggtc tagagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga
     1021 catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg
     1081 tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg catccataca
     1141 ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga cctgcgagca
     1201 gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa
     1261 tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt
     1321 gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa ggaaaagact
     1381 cacgtttcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1441 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1501 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1561 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1621 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggcaaaac agcattccag
     1681 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     1741 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     1801 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     1861 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc
     1921 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     1981 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2041 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2101 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2161 gagtttttct aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg
     2221 tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt
     2281 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     2341 gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc
     2401 atccagtgtc gaaaacgagc tcccgggtac ccagctttct tgtacaaagt ggttggtggt
     2461 ggcggatacc catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat
     2521 gcaggatcct atccatatga cgttccagat tacgcttgaa ctagttggtg tggaagtcct
     2581 aatatcggtg atcaatatag tggttgacat gctggctagt caacattgag ccttttgatc
     2641 atgcaaatat attacggtat tttacaatca aatatcaaac ttaactattg actttataac
     2701 ttatttaggt ggtaacattc ttataaaaaa gaaaaaaatt actgcaaaac agtactagct
     2761 tttaacttgt atcctaggtt atctatgctg tctcaccata gagaatatta cctatttcag
     2821 aatgtatgtc catgattcgc cgggtaaata catataatac acaaatctgg cttaataaag
     2881 tctataatat atctcataaa gaagtgctaa attggctagt gctatatatt tttaagaaaa
     2941 tttcttttga ctaagtccat atcgactttg taaaagttca cattagcata catatattac
     3001 acgagccaga aatagtaact tttgcctaaa tcacaaattg caaaatttaa ttgcttgcaa
     3061 aaggtcacat gcttataatc aactttttta aaaatttaaa atactttttt attttttatt
     3121 tttaaacata aatgaaataa tttatttatt gtttatgatt accgaaacat aaaacctgct
     3181 caagaaaaag aaactgtttt gtccttggaa aaaaagcact acctaggagc ggccaaaatg
     3241 ccgaggcttt catagcttaa actctttaca gaaaataggc attatagatc agttcgagtt
     3301 ttcttattct tccttccggt tttatcgtca cagttttaca gtaaataagt atcacctctt
     3361 agagttcgat gataagctgt caaacatgag aattaattcc acatgttaaa atagtgaagg
     3421 agcatgttcg gcacacagtg gaccgaacgt ggggtaagtg cactagggtc cggttaaacg
     3481 gatctcgcat tgatgaggca acgctaatta tcaacatata gattgttatc tatctgcatg
     3541 aacacgaaat ctttacttga cgacttgagg ctgatggtgt ttatgcaaag aaaccactgt
     3601 gtttaatatg tgtcactgtt tgatattact gtcagcgtag aagataatag taaaagcggt
     3661 taataagtgt atttgagata agtgtgataa agtttttaca gcgaaaagac gataaataca
     3721 agaaaatgat tacgaggata cggagagagg tatgtacatg tgtatttata tactaagctg
     3781 ccggcggttg tttgcaagac cgagaaaagg ctagcaagaa tcgggtcatt gtagcgtatg
     3841 cgcctgtgaa cattctcttc aacaagtttg attccattgc ggtgaaatgg taaaagtcaa
     3901 ccccctgcga tgtatatttt cctgtacaat caatcaaaaa gccaaatgat ttagcattat
     3961 ctttacatct tgttatttta cagattttat gtttagatct tttatgcttg cttttcaaaa
     4021 ggcttgcagg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt
     4081 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac
     4141 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc
     4201 tggcgtcagt ccaccagcta acataaaatg taagctctcg gggctctctt gccttccaac
     4261 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa
     4321 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt
     4381 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga
     4441 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc
     4501 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt
     4561 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct
     4621 attgggcaca catataatac ccagcaagtc agcatcggaa tctagtgcac attctgcggc
     4681 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat
     4741 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag
     4801 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttga
     4861 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4921 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4981 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5041 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5101 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5161 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5221 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5281 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5341 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5401 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5461 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5521 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5581 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5641 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5701 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5761 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5821 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5881 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5941 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6001 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6661 ggggggcgga gcctatggaa a
//
