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GeneCorner plasmid details

Last data update: 21 October 2020 04:26 CEST

Plasmid name: pCAGGSMtCAT (LMBP 3482)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3482.gb (View with Genome Compiler)
p3482.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human manganese superoxide dismutase cDNA (hMnSOD); mitochondrial targeting sequence (MTS)
Rat catalase cDNA (rCAT); fragment (rCATf)
Human catalase cDNA (hCAT); fragment (hCATf)
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; pCAGGShMnSOD; pCMVrhCAT
Further information: The plasmid was constructed as follows: 1) the mitochondrial targeting sequence (MTS) of the human manganese superoxide dismutase gene (hMnSOD) was amplified by PCR on pCAGGShMnSOD; 2) the hybrid rat/human catalase mature sequence was amplified by PCR on pCMVrhCAT (a TGA termination codon was introduced, 48 nucleotides of the coding sequence of hCAT are missing); 3) the MTS sequence and the hybrid catalase sequence were fused by PCR; 4) the fusion gene was inserted as an EcoRI/MstII(SauI) fragment between the EcoRI and MstII(SauI) sites of pCAGGS.
pCAGGSMtCAT is useful for highly efficient expression of MTS-rhCAT 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.
The catalase fragment from the ATG to the PvuII site originates from rat, the fragment from the PvuII site to the termination codon originates from human.
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.
he nucleotide sequence of the human MnSOD cDNA corresponds with the EMBL Nucleotide Sequence Database accession number Y00472.1, with minor corrections in the 5' UTR and with the Ala->Val polymorphism in the MTS. The differences were confirmed by sequence analysis at the Department of Biomedical Molecular Biology (Ghent University, Belgium).
Other name of the plasmid is pCAGGS/Mt-catalase.
EMBL Accession number: Y00472.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 29/03/2006
Sequence detail:
Mitochondrial targeting sequence (MTS) of the human manganese superoxide dismutase cDNA (hMnSOD)fused to the hybrid rat/human catalase (CAT) mature sequence:
    
5'...ATG.TTG.AGC.CGG.GCA.GTG.TGC.GGC.ACC.AGC.AGG.CAG.CTG.GCT.CCG  
     MET Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro 

                                 MTS -->|--> rat CAT
     GTT.TTG.GGG.TAT.CTG.GGC.TCC.AGG.CAG.ATG.GCG.GAC.AGC.CGG.GAC...3' 
     Val Leu Gly Tyr Leu Gly Ser Arg Gln MET Ala Asp Ser Arg Asp
      *

*: Polymorphism (wild type = GCT, Ala).


Primer sequence to introduce the TGA termination codon:

                  <------------ hCATf -------------
5' GCGCCTGAGG.TCA.GGT.GTG.AAT.CGC.ATT.CTT.AGG.CTT.C 3'
      MstII   +++
      SauI

+++: Termination codon.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglII/XbaI, EcoRI/PstI, NcoI/SspI, PvuII/XmnI, SalI and SfiI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr J. Grooten(1)(2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: PhD thesis Vera Goossens (1996)
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:

pCAGGSMtCAT (LMBP 3482) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr J. Grooten and was published in Vera Goossens, 1996.

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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