Last data update: 22 October 2020 04:25 CEST
Plasmid name: pCAGGS-Flag-hABIN1-P299A (LMBP 5132)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1, NAF1); 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-E-hABIN1-P299A; pCAGGS-FLAGmA20|
|Further information:||The plasmid was constructed by ligating the three following fragments: 1) the 2215 bp ScaI/KpnI fragment from pCAGGS-FLAGmA20 2) a 315 bp KpnI/SphI PCR fragment using pCAGGS-E-hABIN1-P299A as template 3) the 4225 bp SphI/ScaI fragment from pCAGGS-E-hABIN1-P299A.
As compared to the wt human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) cDNA, the proline (P) codon at position 299 is replaced by an alanine (A) codon resulting in the loss of an AvaI site and the isoleucine (I) codon at position 9 contains a silent mutation (atc -> att) resulting in the loss of a DpnI and a Sau3AI site.
pCAGGS-Flag-hABIN1-P299A is useful for highly efficient expression of hABIN-1m, fused in phase to the N-terminal FLAG epitope tag, under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.
The AG promoter sequence consists of the chicken β-actin promoter, the first exon and part of the first intron (that seems to have a strong enhancer-like activity) linked to a rabbit β-globin fragment, consisting of a 3' part of the second intron (inclusive a branch point which is required for normal splicing reactions) and a 5' part of the third exon.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of the hABIN-1 cDNA was obtained from Genbank (Accession number NM_006058.2).
Other name of the plasmid is pCAGGS-Flag-hABIN1-FLX.
|EMBL Accession number:||NM_006058.2, view at GenBank|
|Latest sequence update:||03/01/2006|
Primers used to amplify the KpnI-SphI fragment of the hABIN-1 coding sequence: 1 forward: 5' ... GGGGTACC.ATG.GAA.GGG.AGA.GGA.CCG ... 3' KpnI *** NcoI 375 reverse: 5' ... GCCGCTCGAG.TCA.TTC.AAT.CTT.GGA.CTT.GGC ... 3' XhoI +++ 7 9 11 297 299 301 WT (NM_006058): 5' ... TAC.CGG.ATC.TAC.GAC ... GGC.GCA.CCC.GAG.AAG ... 3' Tyr Arg Ile Tyr Asp Gly Ala Pro Glu Lys DpnI AvaI Sau3AI 7 9 11 297 299 301 P299A mutant: 5' ... TAC.CGG.ATT.TAC.GAC ... GGC.GCA.GCC.GAG.AAG ... 3' Tyr Arg Ile Tyr Asp Gly Ala Ala Glu Lys ^ ^ ***: start codon. +++: termination codon. ^: mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by Prof. Dr R. Beyaert(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-hABIN1-P299A (LMBP 5132) is available at BCCM/GeneCorner. This plasmid was deposited by 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.