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載體描述

The pGBKT7 vector expresses proteins fused to amino acids 1–147 of the GAL4 DNA binding domain (DNA-BD). In yeast, fusion proteins are expressed at high levels from the constitutive ADH1 promoter (PADH1); transcription is terminated by the T7 and ADH1 transcription termination signals (TT7 & ADH1). pGBKT7 also contains the T7 promoter, a c-Myc epitope tag, and a MCS. pGBKT7 replicates autonomously in both E. coli andS. cerevisiae from the pUC and 2 μ ori, respectively. The vector carries the Kanr for selection in E. coli and the TRP1 nutritional marker for selection in yeast. Yeast strains containing pGBKT7 exhibit a higher transformation efficiency than strains carrying other DNA-BD domain vectors.

pGBKT7 is the DNA-BD Vector included with Clontech's Matchmaker? Systems. The MCS of pGBKT7 contains unique restriction sites in frame with the 3' end of the GAL4 DNA-BD for constructing fusion proteins with a bait protein. The bait protein is also expressed as a fusion to a c-Myc epitope tag. c-Myc tagged proteins can be identified with antibodies raised to this common epitope, eliminating the need to generate specific antibodies to new proteins. The T7 promoter is used for in vitro transcription and translation of the epitope tagged fusion protein. Note that the DNA-BD is not expressed during the in vitro transcription and translation reactions.

The MCS in pGBKT7 is compatible with those in pMyc-CMV and pHA-CMV, Clontech's epitope tagged mammalian expression vector set (Cat. No. 631604). As a result, the target gene can be shuttled into these vectors in order to confirm protein interactions in vivo.

載體序列

LOCUS       Exported                7303 bp ds-DNA     circular SYN 01-APR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pGBKT7

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 7303)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Monday, Apr 1, 2019 from SnapGene Viewer 4.3.6

FEATURES             Location/Qualifiers

     source          1..7303

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        30..734

                     /gene="S. cerevisiae ADH1"

                     /label=ADH1 promoter

                     /note="promoter for alcohol dehydrogenase 1"

     CDS             762..1202

                     /codon_start=1

                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"

                     /product="DNA binding domain of the GAL4 transcriptional

                     activator"

                     /label=GAL4 DNA binding domain

                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK

                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK

                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"

     promoter        1213..1231

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     primer_bind     1213..1231

                     /label=T7 primer

     CDS             1251..1280

                     /codon_start=1

                     /product="Myc (human c-Myc proto-oncogene) epitope tag"

                     /label=Myc

                     /translation="EQKLISEEDL"

     misc_feature    1281..1333

                     /label=MCS

     terminator      1338..1385

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     terminator      1412..1599

                     /gene="S. cerevisiae ADH1"

                     /label=ADH1 terminator

                     /note="transcription terminator for the S. cerevisiae

                     alcohol dehydrogenase 1 (ADH1) gene"

     primer_bind     complement(1623..1639)

                     /label=M13 rev

                     /note="common sequencing primer, one of multiple similar

                     variants"

     protein_bind    1647..1663

                     /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(1671..1701)

                     /label=lac promoter

                     /note="promoter for the E. coli lac operon"

     protein_bind    1716..1737

                     /label=CAP binding site

                     /bound_moiety="E. coli catabolite activator protein"

                     /note="CAP binding activates transcription in the presence

                     of cAMP."

     rep_origin      complement(2025..2613)

                     /direction=LEFT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     polyA_signal    2942..2989

                     /label=HSV TK poly(A) signal

                     /note="herpes simplex virus thymidine kinase

                     polyadenylation signal (Cole and Stacy, 1985)"

     CDS             complement(3221..4015)

                     /codon_start=1

                     /gene="aph(3')-II (or nptII)"

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /label=NeoR/KanR

                     /note="confers resistance to neomycin, kanamycin, and G418

                     (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     promoter        complement(4016..4120)

                     /gene="bla"

                     /label=AmpR promoter

     rep_origin      4147..5489

                     /label=2u ori

                     /note="yeast 2u plasmid origin of replication"

     protein_bind    complement(5110..5157)

                     /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)."

     promoter        5748..6029

                     /gene="S. cerevisiae TRP1"

                     /label=TRP1 promoter

     CDS             6030..6704

                     /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(6807..7262)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

ORIGIN

        1 cggtgcgggc ctcttcgcta ttacgccaga tccttttgtt gtttccgggt gtacaatatg

       61 gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata ccttcgttgg

      121 tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac cagacaagac

      181 ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg tacataacga

      241 actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca ctaccctttt

      301 tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct ttttttttct

      361 tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaat gatggaagac

      421 actaaaggaa aaaattaacg acaaagacag caccaacaga tgtcgttgtt ccagagctga

      481 tgaggggtat ctcgaagcac acgaaacttt ttccttcctt cattcacgca cactactctc

      541 taatgagcaa cggtatacgg ccttccttcc agttacttga atttgaaata aaaaaaagtt

      601 tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt ttcctcgtca

      661 ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa gctataccaa

      721 gcatacaatc aactccaagc ttgaagcaag cctcctgaaa gatgaagcta ctgtcttcta

      781 tcgaacaagc atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga

      841 agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt

      901 ctccgctgac tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc

      961 tatttctact gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac

     1021 aggatataaa agcattgtta acaggattat ttgtacaaga taatgtgaat aaagatgccg

     1081 tcacagatag attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa

     1141 taagtgcgac atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat

     1201 cgccggaatt tgtaatacga ctcactatag ggcgagccgc catcatggag gagcagaagc

     1261 tgatctcaga ggaggacctg catatggcca tggaggccga attcccgggg atccgtcgac

     1321 ctgcagcggc cgcataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt

     1381 ttttgcgcgc ttgcagccaa gctaattccg ggcgaatttc ttatgattta tgatttttat

     1441 tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt

     1501 aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt

     1561 atagcatgag gtcgctctta ttgaccacac ctctaccggc atgcaagctt ggcgtaatca

     1621 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga

     1681 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt

     1741 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga

     1801 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc

     1861 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg

     1921 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc

     1981 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc

     2041 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga

     2101 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc

     2161 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat

     2221 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg

     2281 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc

     2341 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga

     2401 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact

     2461 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt

     2521 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag

     2581 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg

     2641 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa

     2701 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata

     2761 tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc gagccctggg

     2821 ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc gtattggccc

     2881 caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac cccgcgttta

     2941 tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg

     3001 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg gtgggcgaag

     3061 aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt

     3121 ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg

     3181 ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa

     3241 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc

     3301 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct

     3361 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt

     3421 ccaccatgat attcggcaag caggcatcgc catgagtcac gacgagatcc tcgccgtcgg

     3481 gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt

     3541 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat

     3601 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg

     3661 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc

     3721 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag

     3781 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt

     3841 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca

     3901 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata

     3961 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatactct

     4021 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat

     4081 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc

     4141 cacctgaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta

     4201 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg

     4261 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag

     4321 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga

     4381 gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc

     4441 gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct

     4501 ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta

     4561 ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga

     4621 ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat tatattctat

     4681 accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt

     4741 ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg

     4801 tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg

     4861 tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt

     4921 tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca

     4981 aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt

     5041 acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa

     5101 aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaaa

     5161 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact

     5221 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc

     5281 atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg

     5341 tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca

     5401 gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc

     5461 aatgctatca tttcctttga tattggatca tactaagaaa ccattattat catgacatta

     5521 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt

     5581 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc

     5641 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt

     5701 aactatgcgg catcagagca gattgtactg agagtgcacc atagatcaac gacattacta

     5761 tatatataat ataggaagca tttaatagaa cagcatcgta atatatgtgt actttgcagt

     5821 tatgacgcca gatggcagta gtggaagata ttctttattg aaaaatagct tgtcacctta

     5881 cgtacaatct tgatccggag cttttctttt tttgccgatt aagaattaat tcggtcgaaa

     5941 aaagaaaagg agagggccaa gagggagggc attggtgact attgagcacg tgagtatacg

     6001 tgattaagca cacaaaggca gcttggagta tgtctgttat taatttcaca ggtagttctg

     6061 gtccattggt gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt gctctagatt

     6121 ccgatgctga cttgctgggt attatatgtg tgcccaatag aaagagaaca attgacccgg

     6181 ttattgcaag gaaaatttca agtcttgtaa aagcatataa aaatagttca ggcactccga

     6241 aatacttggt tggcgtgttt cgtaatcaac ctaaggagga tgttttggct ctggtcaatg

     6301 attacggcat tgatatcgtc caactgcatg gagatgagtc gtggcaagaa taccaagagt

     6361 tcctcggttt gccagttatt aaaagactcg tatttccaaa agactgcaac atactactca

     6421 gtgcagcttc acagaaacct cattcgttta ttcccttgtt tgattcagaa gcaggtggga

     6481 caggtgaact tttggattgg aactcgattt ctgactgggt tggaaggcaa gagagccccg

     6541 aaagcttaca ttttatgtta gctggtggac tgacgccaga aaatgttggt gatgcgctta

     6601 gattaaatgg cgttattggt gttgatgtaa gcggaggtgt ggagacaaat ggtgtaaaag

     6661 actctaacaa aatagcaaat ttcgtcaaaa atgctaagaa ataggttatt actgagtagt

     6721 atttatttaa gtattgtttg tgcacttgcc gatctatgcg gtgtgaaata ccgcacagat

     6781 gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc

     6841 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc

     6901 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag

     6961 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga

     7021 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc

     7081 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa

     7141 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt

     7201 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc

     7261 gtccattcgc cattcaggct gcgcaactgt tgggaagggc gat

//