Last data update: 30 November 2020 04:30 CET
Plasmid name: pCAGGS-EmCASP-1prop20m3 (LMBP 4349)
|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); pro-p20 subunit C285S-2DE mutant
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|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
|Further information:||The plasmid was constructed as follows: the mutated pro-p20 fragment (from codon 2 up to and including 295) of the mouse cysteinyl aspartate specific proteinase 1 (mCASP-1) C285S-2DE mutant cDNA was picked up from pCAGGS-EmCASP-1m3 by the use of primers and inserted in frame to the N-terminal E-tag between the NotI and XmaI sites of pCAGGS-EmCASP-1m3.
The mouse caspase-1pro-p20-C285S-2DE coding sequence contains the following point mutations: (i) converting two Asp (D) codons of the prodomain to Glu (E) to destroy potential cleavage sites; (ii) converting the Cys in the active site of the conserved QACRG sequence to Ser (QASRG); and (iii) creating additional BalI (MscI), KpnI and NheI restriction sites.
The Cys conversion is described as C285S, according to the numbering of the codons in the human caspase-1 coding sequence, although the mutated mouse serine is located on position 284.
pCAGGS-EmCASP-1prop20m3 is useful for highly efficient expression of the mutated pro-p20 subunit of mouse caspase-1 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.
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 the wt mCASP-1 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number BC008152.1.
Other name of the plasmid is pCAGGSEprop20casp-1mut.
|EMBL Accession number:||BC008152.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||27/02/2003|
Forward primer (12): 5' CGC.AAG.GAA.GCG.GCC.GCT.GCT.GAC.AAG.ATC.CTG.AGG.GC 3' NotI Reverse primer (13): 5' GT.CGC.CCC.GGG.TTA.TTT.TAA.CAA.CAC.CAC.TCC.TTG.TTT.CTC 3' XmaI Nucleotide sequence of the mutated mouse CASP-1 pro-p20 cDNA: E-tag --------------------------------------------------- 5' … GAATTCCACC ATG.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAC.CCG.CTG.GAA.CCG.CGT.GCG.GCC. Met Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala EcoRI BspMII NotI |--> mouse CASP-1 | 2 3 4 100 103 110 GCT.GCT.GAC.AAG...TTT.GTT.GCT.ACA.GAA.GAA.TCT.AAA.GGT.GGC.CAT.CCT.TCA.TCC.TCA.GAA Ala Ala Asp Lys Phe Val Ala Thr Glu Glu Ser Lys Gly Gly His Pro Ser Ser Ser Glu --* * * E BalI (MscI) prodomain --> | --> p30 120 122| 125 130 ACA.AAG.GAA.GAA.CAG.AAC.AAA.GAA.GAG.GGT.ACC.TTT.CCA.GGA.CTG.ACT.GGG... Thr Lys Glu Glu Gln Asn Lys Glu Glu Gly Thr Phe Pro Gly Leu Thr Gly --* * * E KpnI p20 ->| 280 284 290 295| ...ATT.ATT.CAA.GCT.AGC.CGT.GGA.GAG.AAA.CAA.GGA.GTG.GTG.TTG.TTA.AAA.TAA.CCCGGG Ile Ile Gln Ala Ser Arg Gly Glu Lys Gln Gly Val Val Leu Leu Lys XmaI * * *-- Q A S R G NheI *: mutated nucleotide Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: NcoI and XbaI.|
|History of deposit:||This plasmid was deposited by P. Schotte(1) (2) and 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|
|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)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGS-EmCASP-1prop20m3 (LMBP 4349) is available at BCCM/GeneCorner. This plasmid was deposited by P. Schotte and 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.