Last data update: 21 October 2021 10:13 CEST
Plasmid name: pdcDNA-FLAGMyc (LMBP 4705)
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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)|
FLAG epitope tag; N-terminal
Myc epitope; C-terminal
|Promoter:||Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Phage SP6 promoter
Phage T7 gene 10 promoter (T7g10)
Simian virus 40 early promoter (SV40 early)
Escherichia coli lac operon promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Terminator:||Simian virus 40 polyadenylation signal (SV40 polyA)
Bovine growth hormone polyadenylation signal (BGH polyA)
|Selection marker:||Ampicillin (amp)
Neomycin (neo; G418)
|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:||pcDNA3 was converted to a Gateway destination vector as follows: 1) The Gateway reading frame cassette A (rfA, from Invitrogen) was amplified with a forward primer containing a HindIII site and the FLAG tag, and with a reverse primer containing a Myc epitope and an XhoI site. 2) The PCR product and the vector pcDNA3 (Invitrogen) were digested with HindIII and XhoI and ligated together, resulting in pdcDNA-FLAGMyc.
pdcDNA-FLAGMyc is a Gateway destination vector, containing the ccdB gene of the E. coli F plasmid, flanked by the bacteriophage λ attR recombination sites.
pdcDNA-FLAGMyc is designed for fusing a gene of interest to the N-terminal FLAG epitope tag and C-terminal Myc epitope 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.
This vector has been shown to provide resistance to kanamycin, although growth was reduced compared to growth on medium containing ampicillin.
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 LT727165.1.
The absence of a second enhancer in the SV40 early promoter was shown by sequence analysis.
Other names of the plasmid are pDEST-FLAGMyc and pdcDNAflag-myc.
|EMBL Accession number:||LT727165.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||05/09/2011|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, EcoRV/NotI, EcoRV/XhoI, HindIII and NcoI/XmnI.|
|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
|Related plasmid reference:||Tanghe et al., Cell. Mol. Life Sci. (2018) [PMID: 29435596] [DOI: 10.1007/s00018-018-2763-6]
Bradshaw et al., J. Biol. Chem. 292 (2017), 9583-9598 [PMID: 28438837] [DOI: 10.1074/jbc.M116.767939]
|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. This culture grows very slow: take 24 hours into account for saturated growth!|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pdcDNA-FLAGMyc (LMBP 4705) 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.