Last data update: 24 January 2024 16:39 CET
Plasmid name: pcDNA3-di-RF-HRV (LMBP 5252)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p5252.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Renilla reniformis luciferase gene (rLUC) Photinus pyralis (firefly) luciferase gene (LUC); mutated coding region (LUCm; luc(+)) |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer Phage SP6 promoter Phage T7 gene 10 promoter (T7g10) Simian virus 40 early promoter (SV40 early) Escherichia coli lac operon promoter |
| Ribosome binding site: |
Internal ribosome entry site (IRES) of the human rhinovirus (HRV) |
| 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: | pcDNA3; pXLJ-HRV (10-611); pBluescript-fluc; pSV-Sport-Rluc |
| Further information: | The plasmid was constructed by first cloning the HRV IRES, as a SalI-NcoI fragment from the pXLJ-HRV (10-611) vector, and the LUCm gene, as an NcoI-XbaI fragment of pBluescript-fluc, into the pUC18 plasmid by a three-point ligation. In a second three-point ligation the complete HRV-LUCm insert was cloned as a SalI-EcoRI fragment together with the rLUC gene, as a KpnI-SalI fragment from pSV-Sport-Rluc, in the KpnI-EcoRI linearized pcDNA3 expression plasmid. pcDNA3-di-RF-HRV is a dicistronic expression vector with the internal ribosome entry site (IRES) of the rhinovirus (HRV) in the intercistronic region between upstream rLUC and downstream LUCm coding sequences. The IRES can drive translation of the downstream LUCm sequence independently of the 5'-cap structure bound to the 5'-end of the mRNA molecule. 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 the HRV IRES corresponds with the EMBL Nucleotide Sequence Database accession number DQ875932.2. Other name of the plasmid is di pcDNA3HRV. |
| EMBL Accession number: | DQ875932.2, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 14/12/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AvaI/XmnI, BglII, KpnI/XbaI and NcoI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Y. Bruynooghe(1) (2) and Prof. Dr R. Beyaert(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Tinton et al., Biochem. J. 385 (2005), 155-163 [PMID: 15330758] |
| Related plasmid reference: | Sherf et al., Promega Notes Magazine 49 (1994), 14-21 |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 MC1061 |
| Host reference: | Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Related host reference: | Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pcDNA3-di-RF-HRV (LMBP 5252) is available at BCCM/GeneCorner. This plasmid was deposited by Y. Bruynooghe and Prof. Dr R. Beyaert and was published in Tinton et al., 2005. |
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.