Last data update: 12 August 2022 09:30 CEST
Plasmid name: pDEST-GADT7 (LMBP 4544)
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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 activation domain (GALad)
Influenza HA epitope encoding the haemagglutinin tagging peptide; N-terminal
|Promoter:||Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1)
Phage T7 gene 10 promoter (T7g10)
Escherichia coli lac operon promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Terminator:||Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1)
Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB)
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
Saccharomyces cerevisiae; leu2(-)
|Further information:||The plasmid was constructed by cloning the Gateway reading frame cassette B (rfB, from Invitrogen) between the blunted EcoRI and BamHI sites of pGADT7 (Clontech).
pDEST-GADT7 is a Gateway destination vector, containing the ccdB gene of the E. coli F plasmid, flanked by the bacteriophage λ attR recombination sites.
pDEST-GADT7 is designed for fusing a gene of interest to the N-terminal S. cerevisiae GALad domain and HA 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.
The GALad sequence is composed of the initiation codon, the 2nd and 3rd codon from the SV40 large T-antigen, the nuclear localization signal from the SV40 large T-antigen and the activation domain (codons 768-881) from S. cerevisiae GAL4.
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-GADT7 replicates autonomously in both E. coli and S. cerevisiae from the pMB1 and 2μ ori respectively.
The vector carries the ampicillin resistance gene for selection in E. coli and the LEU2 nutritional marker for selection in yeast.
The pGADT7 part of the plasmid sequence file, which was provided by the depositor, corresponds with the pGADT7 sequence on the Clontech website (http://www.clontech.com/BE/Products/Protein_Interactions_and_Profiling/Yeast_Two-Hybrid/Vectors?sitex=10023:22372:US; 9/12/2011), except for the following nucleotides in the pDEST-GADT7 sequence: 1) an extra A in position 1144; 2) A instead of G in position 5298; 3) G instead of T in position 5348, 4) an extra G in position 5950; 5) G instead of A in position 6945; furthermore, an extra G is present in position 4220 of pGADT7.
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 LT727257.1.
Other name of the plasmid is pdGADT7.
|EMBL Accession number:||LT727257.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||24/03/2003|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BanI, EcoRI, NdeI/XhoI and PvuII.|
|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 + ampicillin (100 μg/ml) + chloramphenicol (25 μg/ml)*|
|Cultivation remark:||*: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin. Cultivate for at least 20 hours.|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pDEST-GADT7 (LMBP 4544) 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.