Last data update: 24 January 2024 16:39 CET
Plasmid name: pRcRSVp65S529A (LMBP 5085)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5085.gb
(View with Genome Compiler) p5085.txt p5085.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human nuclear factor κB cDNA (NF-κB); mutated p65 (RELA) subunit |
| Promoter: | Rous sarcoma virus long terminal repeat (RSV-LTR) Simian virus 40 early promoter (SV40 early) 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) 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: | pRcRSVp65; pGal4-p65S529A |
| Further information: | The plasmid was constructed by inserting the 1613 bp AflII (Bst98I)-BstEII fragment of pGal4-p65S529A into the BstEII-AflII (Bst98I) opened pRcRSVp65 vector. As a result, the serine codon at position 529 of the hNF-κB p65 subunit was replaced by an alanine codon and an additional BsaI (Eco31I) site was created. The plasmid was designed for the expression of the mutated hNF-κB p65 subunit under the control of the RSV-LTR promoter in mammalian cells. 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727082.1. The nucleotide sequence of pRc/RSV was obtained from Invitrogen (http://www.invitrogen.com; Version A). The nucleotide sequence of the hNF-κB p65 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number M62399 .1. The nucleotide sequence of the 3' UTR of the hNF-κB p65 cDNA was retrieved from the pRc/CMV-p65 sequence file provided by S. Plaisance (Dept of Biomedical Molecular Biology, Ghent University, Belgium). |
| EMBL Accession number: | M62399.1, view at EMBL, GenBank, DDBJ LT727082.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 26/10/2005 |
| Sequence detail: | Nucleotide sequence of the hNF-kB p65 subunit:
1 529 784
wild-type: ATG.GAC.GAA.CTG.TTC ... GGC.CTC.CTT.TCA.GGA ... CAG.ATC.AGC.TCC.TAA
Met Asp Glu Leu Phe Gly Leu Leu Ser Gly Gln Ile Ser Ser +++
***
1 529 784
S529A mutant: ATG.GAC.GAA.CTG.TTC ... GGT.CTC.CTT.GCA.GGA ... CAG.ATC.AGC.TCC.TAA
Met Asp Glu Leu Phe Gly Leu Leu Ala Gly Gln Ile Ser Ser +++
*** BsaI
^ ^
***: Start codon.
+++: Termination codon.
^ : Mutated nucleotide.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglI/XhoI, Bst98I/BstEII, Csp45I/StuI, EcoRV/NdeI, HindIII/NotI, KpnI and PvuI/SalI. As compared to the compiled nucleotide sequence, the restriction site analysis pattern revealed that EcoRV only cuts once instead of twice. The EcoRV site at position 615 could not be experimentally confirmed. As the compiled nucleotide sequence has not been adapted accordingly, EcoRV is not indicated as a unique site. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr L. Vermeulen(1) and Prof. Dr G. Haegeman(1). (1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Vermeulen et al., EMBO J. 22 (2003), 1313-1324 [PMID: 12628924] |
| 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 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pRcRSVp65S529A (LMBP 5085) is available at BCCM/GeneCorner. This plasmid was deposited by Dr L. Vermeulen and Prof. Dr G. Haegeman and was published in Vermeulen et al., 2003. |
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.