Last data update: 24 January 2024 16:39 CET
Plasmid name: pEF1crmA-T291R (LMBP 5104)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5104.gb
(View with Genome Compiler) p5104.txt p5104.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Cowpox virus cytokine response modifier A cDNA (crmA); mutated sequence V5 epitope; C-terminal Histidine tag (His-tag); C-terminal |
| Promoter: | Human elongation factor 1α promoter (EF1α) Phage T7 gene 10 promoter (T7g10) Simian virus 40 early promoter (SV40 early) |
| Ribosome binding site: |
- |
| Terminator: | Bovine growth hormone polyadenylation signal (BGH polyA) Simian virus 40 polyadenylation signal (SV40 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 Mammalian cells; SV40 permissive cells |
| Parental clone: | pEF1/V5-HisA |
| Further information: | The plasmid was constructed by inserting the EcoRI fragment, containing the T291R mutant crmA cDNA, in the EcoRI opened pEF1/V5-HisA vector. As a result, the C-terminal V5 epitope and histidine tag are not expressed. pEF1crmA-T291R can be used as a plasmid for negative control. The loss-of-function mutation (T291R) creates an extra NruI restriction site. 18 nucleotides are missing in the 5' UTR. The plasmid is designed for high-level expression of crmA-T291R under control of the hEF1α promoter in mammalian cells. The plasmid carries the ampicillin resistance gene for selection in E. coli and the neomycin resistance gene for selection in mammalian cells. There is uncertainty about the presence of a second enhancer in the SV40 early promoter. The absence of a second enhancer in this promoter may cause bacterial leakage expression of the Tn5 neomycin resistance gene. This would cause transformed E. coli cells to be resistant to kanamycin, although growth should be reduced compared to growth on medium containing ampicillin. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727085.1. The nucleotide sequence of the wild type crmA cDNA corresponds with the EMBL Nucleotide Sequence Database accession number M14217.1. Other name of the plasmid is pEF1/V5-HisACrmAmut. |
| EMBL Accession number: | M14217.1, view at EMBL, GenBank, DDBJ LT727085.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 09/11/2006 |
| Sequence detail: | 290 291 295
Wild type crmA: 5'...GAA.GAG.TAT.ACA.GAA.GCA.GCT.GCA.GCA...3'
Glu Glu Tyr Thr Glu Ala Ala Ala Ala
T291R mutant crmA: 5'...GAA.GAG.TAT.CGC.GAA.GCG.GCT.GCA.GCA...3'
Glu Glu Tyr Arg Glu Ala Ala Ala Ala
*** *
NruI
*: Mutated nucleotide.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BamHI, EcoRI, NdeI and NruI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2). It was constructed by E. Van Damme(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 DH5α |
| Host reference: | Focus 8 (1986), 9 |
| Related host reference: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pEF1crmA-T291R (LMBP 5104) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr P. Vandenabeele . |
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.