Last data update: 21 October 2021 10:13 CEST
Plasmid name: pDEST-GBKT7 (LMBP 4543)
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|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)|
Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd)
Myc epitope; N-terminal
|Promoter:||Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1)
Phage T7 gene 10 promoter (T7g10)
Escherichia coli β-lactamase promoter (amp)
Escherichia coli lac operon promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Ribosome binding site (RBS) of the Escherichia coli ampicillin resistance gene (amp)|
|Terminator:||Phage T7 gene 10 terminator (T7g10)
Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1)
Herpes simplex virus (HSV) thymidine kinase polyadenylation signal (TK polyA)
Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator
|Selection marker:||Chloramphenicol (cam)
Neomycin (neo; kanamycin (kan))
|Replicon:||Escherichia coli plasmid pMB1 origin
Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB)
Phage f1 origin
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
Saccharomyces cerevisiae; trp1(-)
|Further information:||The plasmid was constructed by cloning the Gateway reading frame cassette B (rfB, from Invitrogen) between the blunted EcoRI and NotI sites of pGBKT7 (Clontech).
pDEST-GBKT7 is a Gateway destination vector, containing the ccdB gene of the E. coli F plasmid, flanked by the bacteriophage λ attR recombination sites.
pDEST-GBKT7 is designed for fusing a gene of interest to the N-terminal S. cerevisiae GALbd domain and Myc epitope according to the Gateway Cloning Technology (Invitrogen) and for high-level, constitutive, native expression of this gene in yeast cells under control of the S. cerevisiae ADH1 promoter.
A Gateway entry clone, containing the gene of interest flanked by the λ attL recombination sites, recombines with the attR sites of the destination vector, replacing the ccdB gene. Standard E. coli strains with the unreacted destination vector or with the by-product plasmid retaining the ccdB gene will fail to grow. Thus, this negative selection provides high-efficiency recovery of only the desired clones. The chloramphenicol resistance gene located between the two attR sites permits counterselection.
pDEST-GBKT7 replicates autonomously in both E. coli and S. cerevisiae from the pMB1 and 2μ ori respectively.
The vector carries the kanamycin resistance gene for selection in E. coli and the TRP1 nutritional marker for selection in yeast.
This Gateway destination vector is compatible with the human orfeome library which is also available at BCCM/GeneCorner.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727256.1.
The pGBKT7 part of the plasmid nucleotide sequence file, which was provided by the depositor, corresponds with the pGBKT7 sequence as provided on the Clontech website (http://www.clontech.com/BE/Products/Protein_Interactions_and_ Profiling/Yeast_Two-Hybrid/Vectors?sitex=10023:22372:US; 12/09/2011), except for the following nucleotides in the pDEST-GBKT7 sequence: 1) an extra A in position 410; 2) G instead of C in position 1725; 3) G instead of A in position 2466; 4) T instead of C in position 3450, 5) G instead of A in position 3455; 6) an extra T in position 4886; 7) T instead of C in position 5493; 8) A instead of G in position 6816; furthermore, an extra T is present in position 598 of pGBKT7.
Other name of the plasmid is pdGBKT7.
|EMBL Accession number:||LT727256.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||25/03/2003|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BanII, BstZI, EcoRI and SspI.|
|History of deposit:||This plasmid was deposited by Prof. Dr F. Van Roy(1) (2). It was constructed by Dr B. Janssens(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12xB DB3.1|
|Related host reference:||Bernard et al., J. Mol. Biol. 226 (1992), 735-745 [PMID: 1324324]
|Cultivation medium:||LB-Lennox + chloramphenicol (25 μg/ml) + kanamycin (50 μg/ml)*|
|Cultivation remark:||*: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with kanamycin.|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pDEST-GBKT7 (LMBP 4543) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr F. Van Roy .|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.