Last data update: 24 January 2024 16:39 CET
Plasmid name: pHis-p65f311-551 (LMBP 4652)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4652.gb
(View with Genome Compiler) p4652.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human nuclear factor κB cDNA (NF-κB); fragment of the p65 (RELA) subunit Histidine tag (His-tag); N-terminal Xpress epitope; N-terminal Enterokinase cleavage site (EK); N-terminal |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer Phage T7 gene 10 promoter (T7g10) Simian virus 40 early promoter (SV40 early) |
| 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: | pcDNA3.1/HisA |
| Further information: | The plasmid was constructed by inserting a KpnI-EcoRV PCR fragment, coding for the amino acids 311 up to and including 551 of the p65 subunit of the human nuclear factor κB (NF-κB), into the KpnI-EcoRV opened pcDNA3.1/HisA vector. As a result, the described p65 fragment was fused in phase to the N-terminal histidine tag (His-tag) and Xpress‚ epitope. The plasmid is designed for high-level expression of the fusion protein in mammalian cells, under control of the strong hCMV promoter. 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 LT727146.1. The nucleotide sequence of the hNF-κB p65 coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number M62399.1. Other name of the plasmid is pHis-p65(311-551). |
| EMBL Accession number: | M62399.1, view at EMBL, GenBank, DDBJ LT727146.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 30/10/2003 |
| Sequence detail: | Nucleotide sequence at the start of the fusion gene:
------- His-tag ------>
5' ... TTAAGCTTACC.ATG.GGG.GGT.TCT.CAT.CAT.CAT.CAT.CAT.CAT.GGT.ATG.GCT.AGC
HindIII Met Gly Gly Ser His His His His His His Gly Met Ala Ser
*** NheI
NcoI
StyI
------- Xpress epitope ------->
- EK recogn.site ->
ATG.ACT.GGT.GGA.CAG.CAA.ATG.GGT.CGG.GAT.CTG.TAC.GAC.GAT.GAC.GAT.AAG
Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
KpnI
--------------------------- p65f -------------------------->
312
GTA.CCG.ATC.ATG.AAG.AAG.AGT.CCT.TTC.AGC.GGA.CCC.ACC.GAC.CCC.CGG.CCT ... 3'
Val Pro Ile Met Lys Lys Ser Pro Phe Ser Gly Pro Thr Asp Pro Arg Pro
Nucleotide sequence at the end of the fusion protein:
------------------------- p65f ------------------------>
551
5' ... GCG.GAC.ATG.GAC.TTC.TCA.GCC.CTG.CTG.AGT.CAG.ATC.AGC.TCC.TAA.GGGATATC ... 3'
Ala Asp Met Asp Phe Ser Ala Leu Leu Ser Gln Ile Ser Ser +++ EcoRV
***: Start codon.
+++: Termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BanII, EcoRI, EcoRV/KpnI, HindII, PstI, StuI and XhoI. As compared to the compiled nucleotide sequence, the restriction site analysis pattern revealed that PstI cuts four times instead of three times; the 1684 bp PstI fragment is splitted into two fragments of approximately 1350 bp and 350 bp respectively. The location of the extra PstI site has not been determined; consequently, the compiled nucleotide sequence has not been adapted. |
| 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: | - |
| 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: |
pHis-p65f311-551 (LMBP 4652) is available at BCCM/GeneCorner. This plasmid was deposited by Dr L. Vermeulen and Prof. Dr G. Haegeman. |
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.