Last data update: 12 August 2022 09:30 CEST
Plasmid name: pDEST-MycMAS (LMBP 5025)
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|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)|
Myc epitope; N-terminal
Mitochondrial anchor sequence (MAS); C-terminal
|Promoter:||Simian cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Phage SP6 promoter
|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
|Parental clone:||pDEST-Myc; IppNT/ActaNt-Mito/pUHD|
|Further information:||The plasmid was constructed as follows: 1) The mitochondrial anchor sequence (MAS) from IppNT/ActaNt-Mito/pUHD (a gift from Dr M. Petit, Centrum voor Menselijke Erfelijkheid, KULeuven, Belgium and from Dr E. Friederich, Institut Curie, Paris, France) was amplified with appropriate primers. 2) The PCR product and the vector pDEST-Myc were digested with XhoI, blunted, digested with XbaI and ligated together.
pDEST-MycMAS 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-MycMAS is designed for fusing a gene of interest to the 6 N-terminal Myc epitopes and to the C-terminal mitochondrial anchor sequence (MAS) 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.
Other name of the plasmid is pdCS2-MT-MAS.
|EMBL Accession number:||-|
|Latest sequence update:||01/04/2003|
Primers used to amplify the MAS sequence from lppNt/ActaNt-Mito/pUHD: forward: 5' GGACTCGAG.GGG.AAC.CAT.ACG.ACG.TTA 3' XhoI reverse: 5' GGCGGGTCTAGA.TTA.ATT.ATT.TTT.TCT.TAA.TTG.AAT.A 3' XbaI stop
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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
|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 (34 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-MycMAS (LMBP 5025) 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.