Last data update: 28 October 2020 04:17 CET
Plasmid name: pCAGGS-Flag-IKKalpha-W740A (LMBP 5491)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human NF-κB inhibitor kinase α cDNA (IKKα, CHUK, IKBKA); mutated sequence
FLAG epitope tag; N-terminal
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||pCAGGS-FLAGmA20; pCAGGS|
|Further information:||The plasmid was constructed by ligating the three following fragments: 1) the 2240 bp KpnI-XhoI digested PCR fragment, containing the mutated hIKKα coding sequence; 2) the 2473 bp KpnI-FspI fragment from pCAGGS-FLAGmA20; 3) the 2369 bp XhoI-FspI-ApaI fragment from pCAGGS.
As compared to the wt hIKKα cDNA, the tryptophan (W) codon at position 740 was replaced by an alanine (A) codon, creating an additional PvuII site. Consequently, the mutated hIKKα will not bind to NEMO.
pCAGGS-Flag-IKKalpha-W740A is useful for highly efficient expression of hIKKα-W740A under the control of the AG promoter and the human CMV-IE enhancer in various mammalian cells.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726918.1.
The nucleotide sequence of the hIKKα cDNA corresponds with the Genbank accession number NM_001278.3.
Other name of the plasmid is pCAGGS-Flag-IKKα-W740A.
|EMBL Accession number:||NM_001278.3, view at GenBank
LT726918.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||06/04/2007|
Primers used to amplify hIKKα-W740A: 1 5 forward primer: 5' GGAATTCGGTACC.ATG.GAG.CGG.CCC.CCG 3' EcoRI KpnI *** BglI NcoI SmaI 745 740 reverse primer: 5' CCGCTCGAG.TCA.TTC.TGT.TAA.CCA.GCT.GGC.ATC.AAG.ATT.C 3' XhoI +++ HindII ^ ^^^ PvuII 740 reverse wild-type sequence: ... CCA.ACT.CCA.ATC.AAG ... ***: Start codon. +++: Termination codon. ^ : Mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Alw44I, ApaI/PvuII, KpnI, NcoI/NheI, NotI/XhoI and PvuI/XbaI.
As compared to the compiled nucleotide sequence, the restriction site analysis pattern revealed that ApaI only cuts twice instead of three times. The ApaI site at position 5860 could not be experimentally confirmed.
|History of deposit:||This plasmid was deposited by Dr K. Heyninck(1) (2) and Prof. Dr R. Beyaert(1) (2). It was constructed by M. Kreike(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|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)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGS-Flag-IKKalpha-W740A (LMBP 5491) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. Heyninck and Prof. Dr R. Beyaert .|
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.