Last data update: 04 December 2020 04:17 CET
Plasmid name: pLT-mCASP-1p30 (LMBP 4457)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Mouse cysteinyl aspartate specific proteinase 1 cDNA (caspase-1, CASP-1, ICE, Casp1); p30 subunit|
|Promoter:||Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
|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 created by cloning the mCASP-1 cDNA into the ApaI opened pLT10T3 plasmid.
This is a useful phasmid for mouse CASP-1p30 expression in E. coli under control of the strong and efficiently regulatable λ PL or T7 gene 10 promoter. Transcriptional read-through from these 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 phasmid 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.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene, as well as a cI function: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857]. Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727212.1.
The mCASP-1 cDNA was sequenced by BCCM/GeneCorner and corresponds with the EMBL Nucleotide Sequence Database accession number BC008152.1, except for some nucleotides in the 3' UTR.
Other name of the plasmid is pLT10T3p20p10.
|EMBL Accession number:||BC008152.1, view at EMBL, GenBank, DDBJ
LT727212.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||30/03/2009|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: AvaI, HincII, HindIII and NdeI/XhoI.|
|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
|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.