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GeneCorner plasmid details

Last data update: 24 October 2020 04:12 CEST

Plasmid name: pCAGGS-Flag-IKKalpha-DN (LMBP 5484)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p5484.gb (View with Genome Compiler)
p5484.txt
p5484.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human NF-κB inhibitor kinase α cDNA (IKKα, CHUK, IKBKA); kinase-dead mutant (IKKα-DN)
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
Ribosome
binding site:
-
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 hIKKα-DN 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 lysine (K) codon at position 44 was replaced by a methionine (M) codon, resulting in the loss of a MseI site.
pCAGGS-Flag-IKKalpha-DN is useful for highly efficient expression of hIKKα-DN 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 LT726911.1.
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-DN.
EMBL Accession number: NM_001278.3, view at GenBank
LT726911.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 06/04/2007
Sequence detail:
Primers used to amplify hIKKα:

                                  1               5
forward primer: 5' GGAATTCGGTACC.ATG.GAG.CGG.CCC.CCG 3'
                    EcoRI KpnI   ***       BglI
                              NcoI             SmaI

                                 745             741
reverse primer: 5' CCGCTCGAG.TCA.TTC.TGT.TAA.CCA.ACT.CC 3'
                      XhoI   +++      HindII


Primer used to insert the K44M mutation:

               40              44  45
5' CTT.GAT.CTC.AAA.ATA.GCA.ATT.ATG.TCT.TGT.CGC.CTA.GAG.CT 3'
   Leu Asp Leu Lys Ile Ala Ile Met Ser Cys Arg Leu Glu
                                ^

                               44
wild-type sequence:    ... ATT.AAG.TCT ...
                           Ile Lys Ser 
                            MseI

***: 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.
Class: Recombinant plasmid
Type: Plasmid
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
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:

pCAGGS-Flag-IKKalpha-DN (LMBP 5484) 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.

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