Last data update: 12 September 2023 11:24 CEST
Plasmid name: pdECFP (LMBP 4548)
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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 cyan fluorescent variant (ECFP), 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)|
|Selection marker:||Ampicillin (amp)
|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
|Parental clone:||pBluescriptIIKS-; pECFP-C1|
|Further information:||The plasmid was constructed as follows: 1) the kanamycin resistance gene was removed from pECFP-C1 (Clontech) by cutting with BsaI and ClaI; 2) the ampicillin resistance gene was amplified from a pBluescript vector (Stratagene) and cloned into the blunted pECFP-C1 vector, sense relative to the SV40 early promoter, resulting in pECFP-C1amp; 3) the Gateway reading frame cassette B (rfB, from Invitrogen) was cloned between the blunted BglII and BamHI sites of pECFP-C1amp, resulting in pdECFP.
pdECFP is a Gateway destination vector, containing the ccdB gene flanked by the bacteriophage λ attR recombination sites.
pdECFP is designed for fusing a gene of interest to the C-terminus of the ECFP 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.
ECFP is a cyan fluorescent variant of the wild type GFP that has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = major peak at 433 nm and a minor peak at 453 nm; emission maximum = major peak at 475 nm and a minor peak at 501 nm) and which contains the amino acid substitutions Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to Trp, Asn-146 to Ile, Met-153 to Thr and Val-163 to Ala.
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 LT727259.1.
Nucleotide sequence analysis by BCCM/GeneCorner proved that the ampicillin resistance coding sequence, but not the ampicillin promoter, is inserted antisense relative to the SV40 early promoter. Although a BLAST search didn't allow the identification of a promoter upstream of the ampicillin resistance coding sequence, the construct-carrying E. coli culture proved to grow in the presence of this antibiotic.
Other names of the plasmid are pdECFP-C1amp and pDEST-ECFP.
|EMBL Accession number:||LT727259.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||19/03/2007|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Alw44I, BshNI, EcoRI and Kpn2I.|
|History of deposit:||This plasmid was deposited by Dr S. Wiemann(1).
(1) German Cancer Research Center, Heidelberg, Germany
|Plasmid reference:||Simpson et al., EMBO Rep. 1 (2000), 287-292 [PMID: 11256614]
|Related plasmid reference:||Bradshaw et al., J. Biol. Chem. 292 (2017), 9583-9598 [PMID: 28438837] [DOI: 10.1074/jbc.M116.767939]
Manual of Gateway Cloning Technology (Invitrogen)
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 + 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:
|pdECFP (LMBP 4548) is available at BCCM/GeneCorner. This plasmid was deposited by Dr S. Wiemann and was published in Simpson et al., 2000.|
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.