Last data update: 21 October 2021 10:13 CEST
Plasmid name: pDEST-EGFP (LMBP 4542)
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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)|
Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP), N-terminal
|Promoter:||Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Simian virus 40 early promoter (SV40 early)
Escherichia coli β-lactamase promoter (amp)
Escherichia coli lac operon promoter; mutant (lacUV5)
|Ribosome binding site (RBS) of the Escherichia coli ampicillin resistance gene (amp)|
|Terminator:||Simian virus 40 polyadenylation signal (SV40 polyA)
Herpes simplex virus (HSV) thymidine kinase polyadenylation signal (TK polyA)
|Selection marker:||Neomycin (neo; G418; kanamycin (kan))
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
Mammalian cells; SV40 permissive cells
|Further information:||The plasmid was constructed by cloning the Gateway reading frame cassette A (rfA, from Invitrogen) into the blunted, BamHI opened pEGFP-C2 vector (Clontech).
pDEST-EGFP is a Gateway destination vector, containing the ccdB gene flanked by the bacteriophage λ attR recombination sites.
pDEST-EGFP is designed for fusing a gene of interest to the C-terminus of the EGFP variant of the A. victoria GFP DNA according to the Gateway Cloning Technology (Invitrogen), and for high-level, constitutive, native expression of this gene in mammalian cells under control of the human CMV-IE promoter and enhancer.
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.
EGFP is a red-shifted variant of the wild-type GFP that has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = 488 nm; emission maximum = 507 nm). EGFP encodes the GFPmut1 variant, which contains the amino acid substitutions Phe-64 to Leu and Ser-65 to Thr.
The neomycin resistance cassette, consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the HSV-TK gene, allows stably transfected eukaryotic cells to be selected using G418. The E. coli β-lactamase promoter upstream of this cassette expresses kanamycin resistance in E. coli.
The PstI site and the BssHII site in the neomycin phosphotransferase coding sequence have been removed.
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 LT727255.1.
The nucleotide sequence of the EGFP DNA corresponds with the EMBL Nucleotide Sequence Database accession number U55761.1.
Other name of the plasmid is pdEGFP.
|EMBL Accession number:||U55761.1, view at EMBL, GenBank, DDBJ
LT727255.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||20/03/2003|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Alw44I, Kpn2I, NcoI and PvuII.|
|History of deposit:||This plasmid was deposited by Prof. Dr F. Van Roy(1) (2). It was constructed by B. Janssens(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||PhD thesis Martin et al. (2011)
Lippincott-Schwartz et al., Science 300 (2003), 87-91 [PMID: 12677058]
|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 (30 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-EGFP (LMBP 4542) 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.