Last data update: 24 January 2024 16:39 CET
Plasmid name: pRc/CMV-thioredoxin (LMBP 3452)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p3452.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human ATL-derived factor cDNA homologous to thioredoxin (hADF) |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer Simian virus 40 early promoter (SV40 early) Phage T7 gene 10 promoter (T7g10) Phage SP6 promoter |
| 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: | pRc/CMV |
| Further information: | The plasmid was constructed by cutting pRc/CMV with NotI and XbaI and inserting a NotI-XbaI cDNA fragment containing the hADF gene encoding a human ATL-derived factor homologous to thioredoxin. The cDNA insert was amplified by PCR from a bacterial expression plasmid. The 5'-primer contained a complete NotI site and 18 nucleotides surrounding the start codon of the hADF gene. The 3'-primer contained a complete XbaI site and 10 nucleotides of the 3' end of the hADF gene. This is an expression plasmid for high-level expression of thioredoxin in mammalian cells under control of the human cytomegalovirus promoter and enhancer. The bovine growth hormone polyA signal allows efficient processing and termination of transcription. The plasmid permits the in vitro transcription of hADF sense and antisense RNA transcripts respectively from the T7 and the SP6 promoter. pRc/CMV-thioredoxin contains the replication origin of the single-stranded DNA phage f1 (clockwise orientated), so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage, e.g. M13KO7. 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 hADF gene corresponds with the EMBL Nucleotide Sequence Database (Release 38; Accession number X77584). The exact sequence of the primers was not communicated. |
| EMBL Accession number: | X77584, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 21/10/1996 |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. Schultze-Osthoff(1). (1) Institute of Biochemistry, Albert-Ludwigs-University-Freiburg, Freiburg, Germany |
| Plasmid reference: | - |
| Related plasmid reference: | Tagaya et al., EMBO J. 13 (1994), 2244 [PMID: 8187776] Tagaya et al., EMBO J. 8 (1989), 757-764 [PMID: 2785919] |
| 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: |
pRc/CMV-thioredoxin (LMBP 3452) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. Schultze-Osthoff. |
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.