Last data update: 26 July 2021 12:16 CEST
Plasmid name: pCAGGSEhA20R562A (LMBP 3777)
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|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
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
|Parental clone:||pAS2hA20R562A; pCAGGS; pCAGGS-EmCASP-1; pAS2A20|
|Further information:||This plasmid was constructed via a 4-point ligation of the fragments (i) to (iv). Part of the coding sequence of the human zinc finger protein A20 gene (hA20) was amplified by PCR from pAS2A20, introducing a NotI site at the start codon. The NotI/BamHI fragment from this PCR amplified sequence (i), containing the hA20 N-terminal part, was ligated to the BamHI/SalI fragment from pAS2hA20R562A (ii), containing the mutated hA20 C-terminal part. An E-tag was then ligated to the N-terminus of the hA20 coding sequence as a NotI/PvuI fragment from pCAGGS-EmCASP-1(iii). Finally, the ampicillin resistance gene was restored by ligating the XhoI/PvuI fragment from pCAGGS (iv).
pCAGGSEhA20R562A is useful for highly efficient expression of mutated hA20 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.
The mutated hA20 protein (hA20R562A) does not interact with 14-3-3 proteins.
The plasmid can be used for co-immunoprecitations.
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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727384.1.
Other name of the plasmid is pCAGGSEHuA20R562A.
|EMBL Accession number:||M59465, view at EMBL, GenBank, DDBJ
LT727384.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||30/04/2002|
Nucleotide sequence of the mutated hA20 cDNA: 562 wild type hA20: 5' TCC.TGT.CAC.CAG.CGT.TCC.AAG.TCA GAT 3' Ser Cys His Gln Arg Ser Lys Ser Asp R562A mutant: 5' TCC.TGT.CAC.CAG.GCT.AGC.AAG.TCA.GAT 3' Ser Cys His Gln Ala Ser Lys Ser Asp ** ** NheI
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: HindIII, XhoI and XmnI.|
|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:||De Valck et al., Biochem. Biophys. Res. Commun. 238 (1997), 590-594 [PMID: 9299557]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGSEhA20R562A (LMBP 3777) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert and was published in De Valck et al., 1997.|
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.