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    pGBKT7 Map

    Uploaded by

    Juan Jose Martinez
    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. The vector carries the Kan r for selection in e. Coli and the TRP1 nutritional marker for selection in yeast.

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    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd

    pGBKT7 Map

    Uploaded by

    Juan Jose Martinez
    200 views0 pages
    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. The vector carries the Kan r for selection in e. Coli and the TRP1 nutritional marker for selection in yeast.

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    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. The vector carries the Kan r for selection in e. Coli and the TRP1 nutritional marker for selection in yeast.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    200 views0 pages

    pGBKT7 Map

    Uploaded by

    Juan Jose Martinez
    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. The vector carries the Kan r for selection in e. Coli and the TRP1 nutritional marker for selection in yeast.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 0

    United States/Canada

    800.662.2566
    Asia Pacic
    +1.650.919.7300
    Europe
    +33.(0)1.3904.6880
    Japan
    +81.(0)77.543.6116
    Clontech Laboratories, Inc.
    A Takara Bio Company
    1290 Terra Bella Ave.
    Mountain View, CA 94043
    Technical Support (US)
    E-mail: [email protected]
    www.clontech.com
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    pGBKT7 Vector Information PT3248-5
    Cat. No. 630489
    630443

    (PR8Y2643; published 5 Dec 2008)
    Restriction Map and Multiple Cloning Site (MCS) of pGBKT7. Unique restriction sites are in bold.
    Description:
    The pGBKT7 vector expresses proteins fused to amino acids 1147 of the GAL4 DNA binding
    domain (DNA-BD). In yeast, fusion proteins are expressed at high levels from the constitutive
    ADH1 promoter (P
    ADH1
    ); transcription is terminated by the T7 and ADH1 transcription termi-
    nation signals (T
    T7 &

    ADH1
    ). pGBKT7 also contains the T7 promoter, a c-Myc epitope tag, and a
    MCS. pGBKT7 replicates autonomously in both E. coli and S. cerevisiae from the pUC and
    2 ori, respectively. The vector carries the Kan
    r
    for selection in E. coli and the TRP1 nutritional
    marker for selection in yeast. Yeast strains containing pGBKT7 exhibit a higher transformation
    efciency than strains carrying other DNA-BD domain vectors (1).
    TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG GAA TTT

    GTA ATA CGA CTC ACT ATA GGG CGA GCC GCC ATC ATG GAG GAG CAG AAG CTG ATC TCA GAG GAG GAC CTG
    CAT ATG GCC ATG GAG GCC GAA TTC CCG GGG ATC CGT CGA CCT GCA GCG GCC GCA TAACTAGCATAACCCC
    TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGCGCTTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTT
    ATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAA
    T7 Promoter
    1281

    1212

    1342

    c-Myc Epitope Tag


    T7 Terminator
    EcoR I
    Nde I Nco I S I Sma I/
    Xma I
    BamH I Sal I Pst I
    Matchmaker 5' DNA-BD Vector
    Insert Screening Amplimer
    Matchmaker 3' DNA-BD Vector
    Insert Screening Amplimer
    3' DNA-BD Sequencing Primer
    START
    in vitro
    a.a.
    147
    GAL4 DNA-Binding Domain
    T7 Sequencing Primer
    STOP
    (orf 1)
    STOP
    (orf 2)
    STOP
    (orf 3)
    1430

    1155

    pGBKT7
    7.3 kb

    Kan
    r
    2
    ori
    TRP1
    P
    ADH1
    GAL4
    DNA-BD
    P
    T7
    pUC
    ori
    T
    T7 & ADH1
    c-Myc epitope tag
    MCS
    HindIII
    (738)
    HindIII
    (6544)
    HindIII
    (1606)
    f1
    ori
    pGBKT7 Vector Information
    Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3248-5
    2 Version No. PR8Y2643
    Location of features:
    Truncated S. cerevisiae ADH1 promoter (P
    ADH1
    ): 30736
    GAL4 DNA binding domain (DNA-BD) polypeptide amino acids 1147: 7621202
    T7 RNA polymerase promoter: 12121235
    c-Myc epitope tag: 12481280
    Multiple Cloning Site: 12811334
    Transcription termination signals
    T7 terminator: 13351381
    ADH1 terminator: 14141610
    pUC plasmid replication origin: 18382636
    Kanamycin resistance gene: 41443222
    Yeast 2 replication origin: 41485493
    TRP1 coding sequences
    promoter: 55596755
    gene: 60316705
    f1 bacteriophage origin of replication: 675629
    Location of primers:
    T7 Sequencing Primer: 12131233
    3' DNA-BD Sequencing Primer: 15101487
    Propagation in E. coli:
    Suitable host strains: DH5, DH10 & other general purpose strains
    Selectable marker: plasmid confers resistance to kanamycin (50 g/ml) in E. coli hosts
    E. coli replication origin: pUC
    Copy number: ~500
    Plasmid incompatibility group: pMB1/Col E1
    Propagation in S. cerevisiae:
    Suitable host strains: AH109(MATa), Y187(MAT), Y190(MATa), SFY526(MATa), CG1945(MATa),
    HF7c(MATa)
    Selectable marker: TRP1
    S. cerevisiae origin: 2
    Note: The attached sequence le has been compiled from information in the sequence databases, published literature, and other sources,
    together with partial sequences obtained by Clontech. This vector has not been completely sequenced.
    Reference:
    1. Louret, O. F., et al. (1997) BioTechniques 23: 816819.
    Notice to Purchaser
    Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited
    to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties,
    resold, modied for resale, or used to manufacture commercial products or to provide a service to third parties without written approval
    of Clontech Laboratories, Inc. .
    Clontech, Clontech Logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted otherwise.
    Clontech is a Takara Bio Company. 2008 Clontech Laboratories, Inc.
    Use:
    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 pro-
    teins 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 identied with antibodies raised to this common epitope, eliminating the need to generate
    specic 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 conrm protein interactions in vivo.

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