Last data update: 24 January 2024 16:39 CET
Plasmid name: pCAGGSEhA20S565A (LMBP 3776)
New search | Print data sheet |
Price category: | Cat. 1 (cf. price list) |
Status: | GeneCorner core plasmid |
GeneCorner sequence: |
p3776.gb
(View with Genome Compiler) p3776.pdf |
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: | pAS2hA20S565A; 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 pAS2hA20S565A (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). pCAGGSEhA20S565A 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 (hA20S565A) 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 LT727383.1. Other name of the plasmid is pCAGGSEHuA20S565A. |
EMBL Accession number: | M59465, view at EMBL, GenBank, DDBJ LT727383.1, view at EMBL, GenBank, DDBJ |
Latest sequence update: | 15/05/2002 |
Sequence detail: | Nucleotide sequence of the mutated hA20 cDNA: 565 wild type hA20: 5' CAC.CAG.CGT.TCC.AAG.TCA.GAT.CCC 3' His Gln Arg Ser Lys Ser Asp Pro S565A mutant: 5' CAC.CAG.CGA.TCG.AAG.GCA.GAT.CCC 3' His Gln Arg Ser Lys Ala Asp Pro * * * PvuI |
Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AflIII, XhoI and XmnI. |
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: | 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] [DOI: 10.1016/s0022-2836(83)80284-8] 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) |
Cultivation temperature: | 37°C |
Biosafety level: | L1 |
Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pCAGGSEhA20S565A (LMBP 3776) 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.