pBridge酵母三杂交类质粒

基本信息

启动子:	ADH1,TRP1 ,MET17
复制子:	2μ ori,ori
质粒分类:	酵母系列，酵母三杂交载体
质粒大小:	6526bp
原核抗性:	Amp
筛选标记:	TRP1
克隆菌株:	DH5α
培养条件:	37℃，有氧 LB
表达宿主:	酵母细胞
5'测序引物:	M13R:CAGGAAACAGCTATGACC
3'测序引物:	根据序列设计引物

质粒简介

         pBridgeTM expresses two proteins: a DNA-binding domain fusion, and an additional protein (1–3). pBridge thus allows establishment of three-hybrid systems when used in combination with an activation domain fusion vector and yeast strains from any of CLONTECH's GAL4-based two-hybrid systems (#K1604-1, #K1605-1, #K1612-1). This vector generates a hybrid protein that contains the sequences for the GAL4 DNA-binding domain (DNA-BD; a.a. 1–147) and the sequence cloned into MCS I. The fusion protein is expressed in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (NLS) that are an intrinsic part of the GAL4 DNA-BD (3). An additional gene of interest can be cloned into MCS II which is located downstream of an HA epitope and a second NLS. The resulting fusion protein is conditionally expressed from the MET25 promoter in response to methionine levels in the medium; i.e., it is repressed in the presence of 1 mM methionine and expressed in the absence of methionine (1).
         pBridge is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae. It carries the bla gene (for ampicillin resistance in E. coli) and the TRP1 nutritional marker that allow yeast auxotrophs carrying pBridge to grow on limiting synthetic medium lacking tryptophan. Note: Yeast strain Y187 is a methionine auxotroph; therefore, haploid Y187 harboring pBridge cannot be grown on medium lacking methionine.

质粒图谱

质粒序列

LOCUS       Exported                6526 bp ds-DNA     circular SYN 10-SEP-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 18
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6526)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, September 10, 2016 from SnapGene Viewer 3.1.4
FEATURES             Location/Qualifiers
     source          1..6526
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..1165
                     /note="2u ori"
                     /note="yeast 2u plasmid origin of replication"
     promoter        1447..1551
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             1552..2412
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2583..3171
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      3432..3702
                     /gene="S. cerevisae PGK1"
                     /note="PGK1 terminator"
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     CDS             complement(3828..3848)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(3870..3896)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /note="HA"
                     /translation="YPYDVPDYA"
     promoter        complement(3918..4268)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /note="MET17 promoter"
                     /note="expression is repressed by methionine"
     promoter        4498..4528
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    4536..4552
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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     4560..4576
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4590..4691
                     /gene="S. cerevisiae TRP1"
                     /note="TRP1 promoter"
     CDS             4692..5366
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /note="TRP1"
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     terminator      5419..5606
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 terminator"
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(5653..6093)
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /note="GAL4 DNA binding domain"
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     promoter        complement(6121..6517)
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 promoter"
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt
       61 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt
      121 taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa
      181 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc
      241 tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc
      301 gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg
      361 cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg
      421 tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta
      481 gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat
      541 gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag
      601 aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca
      661 ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa
      721 gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg
      781 agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga
      841 tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc
      901 cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc
      961 tctgaagttc ctatactttc tagctagaga ataggaactt cggaatagga acttcaaagc
     1021 gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt
     1081 tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat
     1141 agtgcgtgtt tatgcttaaa tgcgttatgg tgcactctca gtacaatctg ctctgatgcc
     1201 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     1261 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1321 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     1381 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1441 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1501 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1561 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1621 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1681 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1741 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     1801 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1861 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     1921 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     1981 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2041 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2101 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2161 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2221 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2281 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2341 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2401 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2461 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2521 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2581 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2641 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2701 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2761 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2821 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2881 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2941 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3001 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3061 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3121 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3181 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3241 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3301 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     3361 atacgcaaac gcctctcccc gcgcgttggc cgattcatta atgcaggaag atccgaggcc
     3421 tagctttaac gaacgcagaa ttttcgagtt attaaactta aaatacgctg aacccgaaca
     3481 tagaaatatc gaatgggaaa aaaaaactgc ataaaggcat taaaagagga gcgaattttt
     3541 ttttaataaa aatcttaata atcattaaaa gataaataat agtctatata tacgtatata
     3601 aataaaaaat attcaaaaaa taaaataaac tattatttta gcgtaaagga tggggaaaga
     3661 gaaaagaaaa aaattgatct atcgatttca attcaattca atttatttct tttcggataa
     3721 gaaagcaaca cctggcaatt ccttaccttc caataattcc aaagaagcac caccaccagt
     3781 agagacatgg gagatcagcc cgaagatctt cgggctaatg cggccgccac ctttctcttc
     3841 ttcttaggac tagaaggtcc tcccaggctg gcatagtcag gcacgtcata aggatagcta
     3901 gaagccatat ggcccattgt atggatgggg gtaatagaat tgtatctatg tatctgacga
     3961 ccctgtatta cacgaaggaa atagtagatg aaaagacaag agagcaagaa aaaggaaaaa
     4021 cttcctttct aacagacgct ttacttaacc ttatatatat cttatttttt tcatcgagcg
     4081 tgttcaattg gacaaggtgc caccttttcg acacttcggt tattatgtta cacagttttc
     4141 atgaggatgg cgccttgact aacttaatta gtcatttgcc aaccaccaca gttccccaat
     4201 atcgacagct tcacgtgcca tttgccattt tgcgatcatg tgacctcaag agaaaaagca
     4261 aaaaataatg agagctaagg gattcgaacc cttgcatccg aagatatcag agattttaga
     4321 ggttaaatcc acctaaatct gacgccttaa accactcggc caaactccca ttgtatgttg
     4381 gaagaattag aagctaggcc tcggatcttc ctggcacgac aggtttcccg actggaaagc
     4441 gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact cattaggcac cccaggcttt
     4501 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4561 aggaaacagc tatgacatga ttacgaatta attcggtcga aaaaagaaaa ggagagggcc
     4621 aagagggagg gcattggtga ctattgagca cgtgagtata cgtgattaag cacacaaagg
     4681 cagcttggag tatgtctgtt attaatttca caggtagttc tggtccattg gtgaaagttt
     4741 gcggcttgca gagcacagag gccgcagaat gtgctctaga ttccgatgct gacttgctgg
     4801 gtattatatg tgtgcccaat agaaagagaa caattgaccc ggttattgca aggaaaattt
     4861 caagtcttgt aaaagcatat aaaaatagtt caggcactcc gaaatacttg gttggcgtgt
     4921 ttcgtaatca acctaaggag gatgttttgg ctctggtcaa tgattacggc attgatatcg
     4981 tccaactgca tggagatgag tcgtggcaag aataccaaga gttcctcggt ttgccagtta
     5041 ttaaaagact cgtatttcca aaagactgca acatactact cagtgcagct tcacagaaac
     5101 ctcattcgtt tattcccttg tttgattcag aagcaggtgg gacaggtgaa cttttggatt
     5161 ggaactcgat ttctgactgg gttggaaggc aagagagccc cgaaagctta cattttatgt
     5221 tagctggtgg actgacgcca gaaaatgttg gtgatgcgct tagattaaat ggcgttattg
     5281 gtgttgatgt aagcggaggt gtggagacaa atggtgtaaa agactctaac aaaatagcaa
     5341 atttcgtcaa aaatgctaag aaataggtta ttactgagta gtatttattt aagtattgtt
     5401 tgtgcacttg ccggcatgcc ggtagaggtg tggtcaataa gagcgacctc atgctatacc
     5461 tgagaaagca acctgaccta caggaaagag ttactcaaga acaagaattt tcgttttaaa
     5521 acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta tttaataata
     5581 aaaatcataa atcataagaa attcgcccgg aattagcttg gctgcaggtc gacggatccc
     5641 cgggaattcc ggcgatacag tcaactgtct ttgacctttg ttactactct cttccgatga
     5701 tgatgtcgca cttattctat gctgtctcaa tgttagaggc atatcagtct ccactgaagc
     5761 caatctatct gtgacggcat ctttattcac attatcttgt acaaataatc ctgttaacaa
     5821 tgcttttata tcctgtaaag aatccatttt caaaatcatg tcaaggtctt ctcgaggaaa
     5881 aatcagtaga aatagctgtt ccagtctttc tagccttgat tccacttctg tcagatgtgc
     5941 cctagtcagc ggagaccttt tggttttggg agagtagcga cactcccagt tgttcttcag
     6001 acacttggcg cacttcggtt tttctttgga gcacttgagc tttttaagtc ggcaaatatc
     6061 gcatgcttgt tcgatagaag acagtagctt catctttcag gaggcttgct tcaagcttgg
     6121 agttgattgt atgcttggta tagcttgaaa tattgtgcag aaaaagaaac aaggaagaaa
     6181 gggaacgaga acaatgacga ggaaacaaaa gattaataat tgcaggtcta tttatacttg
     6241 atagcaagac agcaaacttt tttttatttc aaattcaagt aactggaagg aaggccgtat
     6301 accgttgctc attagagagt agtgtgcgtg aatgaaggaa ggaaaaagtt tcgtgtgctt
     6361 cgagataccc ctcatcagct ctggaacaac gacatctgtt ggtgctgtct ttgtcgttaa
     6421 ttttttcctt tagtgtcttc catcattttt ttgtcattgc ggatatggtg agacaacaac
     6481 gggggagaga gaaaagaaaa aaaaagaaaa gaagttgcat gcaagc