Last data update: 16 January 2021 04:19 CET
Plasmid name: pCAGGS-E-hA20-C775A-C779A (LMBP 6569)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Depositor's sequence:||not available|
Human TNF alpha induced protein 3 cDNA (TNFAIP3, A20, OTUD7C, GeneID 7128); 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
|Further information:||The point mutation was introduced by an overlap-PCR on pCAGGSEhA20. The construct was digested with DraIII-XhoI and ligated into pCAGGSEhA20 digested with DraIII-XhoI.
pCAGGS-E-hA20-C755A-C779C has 2 point mutations to disrupt Zinc Finger 7. This Zinc finger is thought to have a ubiquitin binding capacity and is needed for NF-kB inhibition.
The plasmid is useful for highly efficient expression of the fusion gene under the control of the AG promoter 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 lacIq strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
|EMBL Accession number:||-|
|Latest sequence update:||22/01/2010|
|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
|Plasmid reference:||Verhelst et al., EMBO J. 31 (2012), 3845-3855 [PMID: 23032186] [DOI: 10.1038/emboj.2012.240]
|Related plasmid reference:||Natoli et al., J. Biol. Chem. 273 (1998), 31262-31272 [PMID: 9813034]
Lin et al., J. Biol. Chem. 281 (2006), 2095-2130 [PMID: 16306043]
Li et al., Biochim. Biophys. Acta 1783 (2008), 1140-1149 [PMID: 18329387]
Li et al., Biochim. Biophys. Acta 1793 (2009), 346-353 [PMID: 18952128]
Afonina et al., EMBO Rep. 17 (2016), 914-927 [PMID: 27113748] [DOI: 10.15252/embr.201642109]
|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-hA20-C775A-C779A (LMBP 6569) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert and was published in Verhelst et al., 2012.|
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.