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GeneCorner plasmid details

Last data update: 24 October 2020 04:12 CEST

Plasmid name: pCAGGS-E-hMKP1-P142H-P360R (LMBP 5140)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p5140.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human MAP (mitogen-activated protein) kinase phosphatase 1 (hMKP-1, hDUSP1); 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: pFlag-hMKP-1; pCAGGS-E-hABIN-1
Further information: The plasmid was constructed by ligating the three following fragments: 1) the 4767 bp BglII/NotI fragment from pCAGGS-E-hABIN-1 2) a 226 bp NotI fragment of pFlag-hMKP-1 and 3) the 901 bp NotI/BglII fragment from pFlag-hMKP-1.
As compared to the wild type human DUSP1 coding sequence, codon 142 (CCC, proline) was replaced by a histidine (CAC) codon, resulting in the loss of an AvaI site and codon 360 (CCC, proline) was replaced by an arginine (CGC) codon. Additionally, the coding sequence includes the following silent nucleotide substitutions: C600A, A780G and G933A. Numbering includes the starting codon, which is absent in this plasmid.
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/tac/trc/N25/O2 promoter it may be advisable to use lacIq strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
Other name of the plasmid is pCAGGS-E-hMKP1.
EMBL Accession number: NM_004417, view at GenBank
Latest sequence update: 04/01/2006
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: -
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-hMKP1-P142H-P360R (LMBP 5140) 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.

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