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GeneCorner plasmid details

Last data update: 30 November 2020 04:30 CET

Plasmid name: pCAGGS-EmCASP-1prop20m3 (LMBP 4349)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4349.gb (View with Genome Compiler)
p4349.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse cysteinyl aspartate specific proteinase 1 cDNA (caspase-1, CASP-1, ICE, Casp1); pro-p20 subunit C285S-2DE mutant
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: pCAGGS-EmCASP-1m3
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
Sequence detail:
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.
Class: Recombinant plasmid
Type: Plasmid
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
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-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.

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