Last data update: 29 October 2020 04:15 CET
Plasmid name: pCAGGS-E-hABIN1-N (LMBP 5128)
|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 N-terminal fragment
|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
|Further information:||The plasmid was constructed by inserting a 1136 bp NotI-XhoI PCR fragment, containing the codons 1 up to and including 375 of the mutated human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) coding sequence, into the NotI-XhoI opened pCAGGS-E-hABIN1-P299A vector.
As compared to the wt hABIN-1 cDNA, the valine (V) codon at position 277 is replaced by an alanine (A) codon creating an additional MspA1I site, the proline (P) codon at position 299 is also 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-E-hABIN1-N is useful for highly efficient expression of the mutated N-terminal fragment of hABIN-1, 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.
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).
|EMBL Accession number:||NM_006058.2, view at GenBank|
|Latest sequence update:||21/12/2005|
Primers used to amplify the mutated N-terminal fragment of the hABIN-1 coding sequence: 1 5 forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAA.GGG.AGA.GGA.CCG ... 3' BamHI NotI *** 375 370 reverse: 5' ... GCCGCTCGAG.TCA.TTC.AAT.CTT.GGA.CTT.GGC ... 3' XhoI +++ 9 277 299 WT (NM_006058): 5' ... CGG.ATC.TAC ... GCA.GTG.GCT ... GCA.CCC.GAG ... 3' Arg Ile Tyr Ala Val Ala Ala Pro Glu DpnI AvaI Sau3AI 9 277 299 V277A-P299A mutant: 5' ... CGG.ATT.TAC ... GCA.GCG.GCT ... GCA.GCC.GAG ... 3' Arg Ile Tyr Ala Ala Ala Ala Ala Glu ^ MspA1I ^ ^ ***: 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-E-hABIN1-N (LMBP 5128) 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.