Last data update: 04 December 2020 04:17 CET
Plasmid name: pLT-mCASP-3p30-C163A (LMBP 9336)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|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
|Promoter:||Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Phage λ major leftward promoter (λ PL)
|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|
|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.|
|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
|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)|
|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.