Last data update: 24 January 2024 16:39 CET
Plasmid name: pCDM8-PKR (LMBP 4513)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4513.gb
(View with Genome Compiler) p4513.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human eukaryotic translation initiation factor 2 alpha kinase 2 cDNA (EIF2AK2, PRKR, PKR, PPP1R83, GeneID 5610) |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
- |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Suppressor tRNA gene (supF; requires a p3 containing host) |
| Replicon: | Bacteriophage M13 origin Escherichia coli plasmid pMB1 origin Polyoma virus origin Simian virus 40 bidirectional origin (SV40) |
| Host range: | Escherichia coli; use strains with the p3 helper plasmid (kan resistant, amber codon in amp and tet) Mammalian cells; SV40 permissive cells |
| Parental clone: | pCDM8-His-PKR |
| Further information: | The plasmid was constructed as follows: (i) pCDM8-His-PKR was digested with NdeI, filled in with Klenow DNA polymerase and a BstXI-EcoRI adaptor/linker (Invitrogen) was ligated; (ii) the 1690 bp BstXI fragment, containing the human PKR cDNA, was then inserted into the BstXI opened pCDM8 vector. Overexpression of human PKR inhibits translation. The T7 promoter is used for in vitro transcription of the cDNA insert. The small HIV (human immunodeficiency virus) fragment (29 nucleotides) between the CMV and T7 promoter sequences has an enhancer function. The SV40 origin of replication permits episomal replication in mammalian cells expressing SV40 large T-antigen (e.g. COS cells). The presence of the polyoma origin of replication enables the vector to replicate episomally in mammalian cells expressing polyoma large T-antigen (e.g. WOB cells). The supF tRNA suppressor gene is used for selection in E. coli (MC1061[p3]). The M13 origin is useful for synthesis of single-stranded DNA upon infection with the M13 helper phage. The SspI fragment (153 bp) of the 3' UTR region of the SV40 small t-antigen is missing, but without effect on the function of the vector. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727247.1. The nucleotide sequence of the human PKR coding sequence corresponds with the EMBL Nucleotide Sequence database accession number M35663.1. The borders of the hPKR insert have been sequenced. Other name of the plasmid is pCDM8-hPKR. |
| EMBL Accession number: | M35663.1, view at EMBL, GenBank, DDBJ LT727247.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 24/02/2003 |
| Sequence detail: | BstXI-EcoRI adaptor (Invitrogen)
5' GAATTCCACCACA 3'
3' CTTAAGGTG 5'
EcoRI
BstXI |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: NcoI, SacI and ScaI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by 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 MC1061[p3] |
| Host reference: | - |
| Related host reference: | Instruction Manual (Invitrogen) Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Helper plasmid: | p3 |
| Cultivation medium: | LB-Lennox + ampicillin (50 μg/ml) + kanamycin (50 μg/ml) + tetracycline (10 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pCDM8-PKR (LMBP 4513) is available at BCCM/GeneCorner. This plasmid was deposited by 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.