Last data update: 12 September 2023 11:24 CEST
Plasmid name: pdECFP (LMBP 4548)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4548.gb
(View with Genome Compiler) p4548.txt p4548.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
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: |
Ribosome binding site (RBS) of the Escherichia coli ampicillin resistance gene (amp) |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Ampicillin (amp) Chloramphenicol (cam) |
| 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. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| 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 |
| Host reference: | - |
| 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 temperature: | 37°C |
| Biosafety level: | L1 |
| 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.