Last data update: 24 January 2024 16:39 CET
Plasmid name: pCAGGS-E-hA20-C624A-C627A (LMBP 6563)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | not available |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human TNF alpha induced protein 3 cDNA (TNFAIP3, A20, OTUD7C, GeneID 7128); mutated sequence E-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-E-hA20-C-C624A-C627A |
| Further information: | pCAGGS-EhA20-C-C624A-C627A was digested with BstXI resulting in a fragment of 701 bp containing the mutations and a fragment of 5414 bp. pCAGGSEhA20 was also digested with BstXI resulting in fragments of 739 and 6539 bp. The 6539 bp fragment was cipped and ligated to the 701 bp fragment, resulting in pCAGGS-E-hA20-C624A-C627A. A20-C624A-C627A contains 2 point mutations (C624A - C627A) in the catalytic site of ZF4 in order to abolish the ubiquitination activity of A20 on RIP1. 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. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| 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: | Afonina et al., EMBO Rep. 17 (2016), 914-927 [PMID: 27113748] [DOI: 10.15252/embr.201642109] Wertz et al., Nature 430 (2004), 694-699 [PMID: 15258597] |
| 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-E-hA20-C624A-C627A (LMBP 6563) 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.