Last data update: 29 October 2020 04:15 CET
Plasmid name: pCAGGS-E-hABIN-1-P299A (LMBP 5127)
|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
|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; pCAGGSEhA20|
|Further information:||The plasmid was constructed by ligating the three following fragments: 1) the 2517 bp XhoI/PvuI fragment from pCAGGS 2) the 2336 bp PvuI/NotI fragment from pCAGGShA20. 3) a 1919 bp NotI/XhoI PCR fragment, containing the human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) mutated sequence.
pCAGGS-E-hABIN1-P299A is useful for highly efficient expression of hABIN-1-P299A, fused in phase to the N-terminal E-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.
As compared to the wt human ABIN-1 cDNA, the 'C' nucleotide at position 895 of the coding sequence has been replaced by a 'G', resulting in a P299A mutation and the loss of an AvaI site
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 human ABIN-1-P299A coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number AJ011895.1, except for amino acid 9 (isoleucine) which is encoded by the wt sequence 'ATC' in the EMBL record and by the silent mutation 'ATT' in the plasmid sequence, resulting in the loss of a DpnI and a Sau3AI site. The P299A mutation is present in the EMBL record.
Other names of the plasmid are pCAGGS-E-hABIN1-FLX and pCAGGS-E-hABIN1-P299A.
|EMBL Accession number:||AJ011895.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||29/10/2010|
Primers used to amplify the hABIN-1 coding sequence: forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAA.GGG.AGA.GGA.CCG ... 3' BamHI NotI *** reverse: 5' ... GCCGCTCGAG.TCA.CTG.AGG.CCC.CTC.ACG ... 3' XhoI +++ 7 9 11 297 299 301 WT: 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:||Restriction enzyme pattern analysed at GeneCorner: AvaI, DpnI, EcoRI, NotI/XhoI, PstI and PvuI.|
|History of deposit:||This plasmid was deposited by Prof. Dr R. Beyaert(1) (2). It was constructed by M. Haegman(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-E-hABIN-1-P299A (LMBP 5127) 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.