Last data update: 24 January 2024 16:39 CET
Plasmid name: pEF6-E-hPKR-KD-K296R (LMBP 4504)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4504.gb
(View with Genome Compiler) p4504.txt p4504.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human eukaryotic translation initiation factor 2 alpha kinase 2 cDNA (EIF2AK2, PRKR, PKR, PPP1R83, GeneID 5610); mutated caspase-generated kinase domain (PKR-KD-K296R) E-tag; N-terminal Myc 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) Synthetic prokaryotic EM7 promoter Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Bovine growth hormone polyadenylation signal (BGH polyA) Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Ampicillin (amp) Blasticidin (bsd) |
| Replicon: | Escherichia coli plasmid pMB1 origin Phage f1 origin Simian virus 40 bidirectional origin (SV40) |
| Host range: | Escherichia coli; preferably recombination-deficient and endonuclease A deficient strains Mammalian cells; SV40 permissive cells |
| Parental clone: | pEF6-hPKR-K296R-MycHis; pEF6/E-Myc-HisA |
| Further information: | The plasmid was constructed by inserting the mutated caspase-generated kinase domain (codon 252 up to 551) of the human PKR cDNA, PKR-KD-K296R, amplified by PCR from pEF6-hPKR-K296R-MycHis, as a NotI/XbaI fragment between the NotI and XbaI sites of pEF6/E-Myc-HisA. The human PKR-KD-K296R fragment is in frame with the N-terminal E-tag. As the human PKR-KD-K296R fragment contains a termination codon, the C-terminal Myc epitope and histidine tag (His-tag) are not expressed. Overexpression of the catalytically inactive, human PKR-KD-K296R does not inhibit translation. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727238.1. The nucleotide sequence of the wild type human PKR coding sequence corresponds with the EMBL Nucleotide Sequence database accession number M35663.1. Other names of the plasmid are pEF6-E-Tag-PKR-KDcaspK296R, pEF6A-E-tag-PKR-KDcasp K296R and pEF6A-E-PKR-KD-K296R. |
| EMBL Accession number: | M35663.1, view at EMBL, GenBank, DDBJ LT727238.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 26/05/2005 |
| Sequence detail: | Primers used for PCR on pEF6-hPKR-K296R-MycHis:
252 260
forward primer: 5' AAGGGCGGCCGCT.ATG.AAA.GAA.ACA.AAG.TAT.ACT.GTG.GAC 3'
NotI
551 550
reverse primer: 5' GGTCTAGA.CTA.ACA.TGT.GTG.TCG.TTC 3'
XbaI ***
Nucleotide sequence of the mutated hPKR cDNA:
296
wild type hPKR: 5' ... GTT.ATT.AAA.CGT.GTT ... 3'
Val Ile Lys Arg Val
K296R hPKR mutant: 5' ... GTT.ATT.CGA.CGT.GTT ... 3'
Val Ile Arg Arg Val
Punctuation indicates reading frame.
***: termination codon. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglII, PstI, XhoI and XmnI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr X. Saelens(1) (2), Dr M. Kalai(1) (2) and Prof. Dr P. Vandenabeele(1) (2) (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Saelens et al., J. Biol. Chem. 276 (2001), 41620-41628 [PMID: 11555640] |
| Related plasmid reference: | Kalai et al., Cell Death Differ. 14 (2007), 1050-1059 [PMID: 17318221] |
| 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: |
pEF6-E-hPKR-KD-K296R (LMBP 4504) is available at BCCM/GeneCorner. This plasmid was deposited by Dr X. Saelens , Dr M. Kalai and Prof. Dr P. Vandenabeele Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium and was published in Saelens et al., 2001. |
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.