Last data update: 24 January 2024 16:39 CET
Plasmid name: pEF6-hPKR-D251N-MycHis (LMBP 4508)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4508.gb
(View with Genome Compiler) p4508.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human eukaryotic translation initiation factor 2 alpha kinase 2 cDNA (EIF2AK2, PRKR, PKR, PPP1R83, GeneID 5610); mutated sequence 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-MycHis; pEF6/Myc-HisC |
| Further information: | The human PKR coding sequence was PCR amplified from pEF6-hPKR-MycHis by the use of mutator primers. As a result, the aspartic acid codon 251 was replaced by an asparagine codon (PKR-D251N). The mutated amplicon was then digested with BamHI and NotI and inserted into the BamHI/NotI opened pEF6/Myc-HisC vector, in frame with a C-terminal Myc epitope and polyhistidine tag (His-tag). Overexpression of human PKR-D251N may inhibit translation. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727242.1. The nucleotide sequence of the wild type human PKR coding sequence corresponds with the EMBL Nucleotide Sequence database accession number M35663.1. Other name of the plasmid is pEF6PKRD251Nmychis. |
| EMBL Accession number: | M35663.1, view at EMBL, GenBank, DDBJ LT727242.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 24/05/2005 |
| Sequence detail: | Primers (251) used for overlap overlap fusion PCR:
forward primer : 5' AC.CTT.CCT.AAC.ATG.AAA.GAA.AC 3'
reverse primer : 5' GT.TTC.TTT.CAT.GTT.AGG.AAG.GT 3'
Nucleotide sequence of the mutated hPKR cDNA:
251
wild type hPKR: 5' ... CTT.CCT.GAC.ATG.AAA.GAA ... 3'
Leu Pro Asp Met Lys Glu
D251N hPKR mutant: 5' ... CTT.CCT.AAC.ATG.AAA.GAA ... 3'
Leu Pro Asn Met Lys Glu
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: NcoI, XhoI and XmaI. |
| 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] |
| 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-hPKR-D251N-MycHis (LMBP 4508) is available at BCCM/GeneCorner. This plasmid was deposited by Dr X. Saelens , Dr M. Kalai and Prof. Dr P. Vandenabeele 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.