Last data update: 09 March 2021 04:23 CET
Plasmid name: pdEYFP (LMBP 4547)
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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 yellow-green fluorescent variant (EYFP), C-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:||Gentamicin (Gm)
|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:||pEYFP-N1; placUV5-gen|
|Further information:||The plasmid was constructed as follows: 1) the kanamycin resistance gene was removed from pEYFP-N1 (Clontech) by cutting with BsaI and ClaI; 2) the gentamicin resistance gene under control of the lacUV5 promoter, amplified from placUV5-gen (Invitrogen), was cloned into the blunted pEYFP-N1 vector, sense relative to the SV40 early promoter, resulting in pEYFP-N1gen; 3) the Gateway reading frame cassette B (rfB, from Invitrogen) was cloned between the blunted XhoI and AgeI sites of pEYFP-N1gen, resulting in pdEYFP.
pdEYFP is a Gateway destination vector, containing the ccdB gene flanked by the bacteriophage λ attR recombination sites.
pdEYFP is designed for fusing the EYFP variant of the A. victoria GFP DNA to the C-terminal end of a gene of interest 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.
EYFP is a yellow-green fluorescent variant of the wild-type GFP that has been optimized for brighter fluorescence and higher expression in mammalian cells (excitation maximum = 513 nm; emission maximum = 527 nm). EYFP encodes the GFP-10C variant, which contains the amino acid substitutions Ser-65 to Gly, Val-68 to Leu, Ser-72 to Ala and Thr-203 to Tyr.
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 LT727258.1.
The area of the gentamycin resistance gene and of the lacUV5 promoter were sequenced by BCCM/GeneCorner.
Other names of the plasmid are pdEYFP-N1gen and pDEST-EYFP.
|EMBL Accession number:||LT727258.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||19/03/2007|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: AflIII, BshNI, HincII and NheI.|
|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 Reports 1 (2000), 287-292 [PMID: 11256614]
|Related plasmid reference:||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) + gentamicin (10 μg/ml)*|
|Cultivation remark:||*: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with gentamycin.|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pdEYFP (LMBP 4547) 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.