Last data update: 21 October 2021 10:13 CEST
Plasmid name: pDEST-Etag (LMBP 4540)
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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)|
|Promoter:||Simian cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Phage SP6 promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Terminator:||Simian virus 40 polyadenylation signal (SV40 polyA)|
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
|Further information:||The plasmid was constructed by ligating the Gateway reading frame cassette C (rfC, from Invitrogen) into the StuI opened pCS2-ET vector. The XbaI site can be used to screen the orientation.
pDEST-Etag is a Gateway destination vector, containing the lethal B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB) flanked by the bacteriophage λ attR recombination sites.
pDEST-Etag is designed for fusing a gene of interest to the N-terminal E-tag according to the Gateway Cloning Technology (Invitrogen) and for high-level, constitutive, native expression of this gene in a wide variety of mammalian and avian cells under control of the strong simian cytomegalovirus immediate early promoter (CMV-IE) 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.
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 LT727253.1.
Other names of the plasmid are pdCS2-ET and pDEST-pCS2+ET.
|EMBL Accession number:||LT727253.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||01/04/2003|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: DraI, HincII, SmaI and XbaI.|
|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:||Manual of Gateway Cloning Technology, Invitrogen
|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.|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pDEST-Etag (LMBP 4540) 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.