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LMBP plasmid details

Last data update: 23 September 2020 04:27 CEST

Plasmid name: pLT-mCASP-3p30-C163A (LMBP 9336)

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

-

Cloned DNA: Mouse caspase 3 cDNA (Casp3, AC-3, Apopain, Caspase-3, CC3, CPP32, mldy, Yama, GeneID 12367); mutated fragment
Histidine tag (His-tag); N-terminal
Enterokinase cleavage site (EK); N-terminal
Strep-tag; C-terminal
Promoter: Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Phage λ major leftward promoter (λ PL)
Ribosome
binding site:
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)
Terminator: Phage fd terminator
Phage T7 gene 10 terminator (T7g10)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli; use strains with a cI function, cIts for PL controlled expression
Parental clone: pLT-mCASP-3p30
Further information: The plasmid was constructed by introducing a C163A mutation into the mouse Casp3 coding sequence of pLT-mCASP-3p30.
This plasmid contains a C163A mutant of the p30 precursor of mouse Casp3 cDNA.
The plasmid can be used to make in vitro translated p30 forms of the inserted caspase.
The H6EK linker is composed of a hexameric histidine-encoding fragment (allowing the synthesis of affinity-tagged fusion proteins) followed by the enterokinase recognition site (allowing the precise release of the mature heterologous gene). This H6EK linker directly follows the ATG start codon.
Transcriptional read-through from the promoters is minimized by the presence of a duplicated T7 transcription terminator and a duplicated fd transcription terminator. Read-through transcription from other plasmid promoters is minimized by the clockwise orientation of the PL and T7 promoters relative to the anticlockwise orientation of the replication origin.
Furthermore, the plasmid is provided with an antisense phage T3 promoter. It also contains the replication origin of the single-stranded DNA phage f1, so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage, e.g. M13KO7.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3). Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
EMBL Accession number: -
Latest sequence update: 10/08/2017
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: The plasmid was deposited by Prof. Dr W. Declercq(1)(2).
(1) Center for Inflammation Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Schotte et al., Biochem. Biophys. Res. Commun. 251 (1998), 379-387 [PMID: 9790964]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061(λ)
Host reference: Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
Other culture collection numbers: -

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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