Last data update: 27 October 2020 04:27 CET
Plasmid name: pCAGGSrhCAT (LMBP 3481)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||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
|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; pCMVrhCAT|
|Further information:||The plasmid was constructed by inserting the hybrid rat-human catalase cDNA as a NcoI/HindIII fragment from pCMVrhCAT into the EcoRI digested pCAGGS vector (all sites were filled in with T4 polymerase).
The fragment from the ATG to the PvuII site originates from rat, the fragment from PvuII to the termination codon originates from the human catalase gene.
pCAGGSrhCAT is useful for highly efficient expression of the hybrid rat-human catalase 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727343.1.
Other name of the plasmid is pCAGGS/r-h-catalase.
|EMBL Accession number:||LT727343.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||06/09/2001|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BamHI, EcoRI, EcoRI/PstI, EcoRI/XhoI, PstI, PvuII/XmnI and XhoI.
Although we could determine non-intensive bands on the agarose gel corresponding to the expected fragments for the EcoRI/PstI and EcoRI/XhoI digestions, the presence of the EcoRI site remains questionable. Theoretically, the EcoRI site should be present, being the result of the EcoRI/NcoI ligation after filling-in the two sites. However, the sequence analysis results of the Department of Molecular Biology (Ghent University, Belgium) revealed the nucleotide sequence 'GAATC' instead of 'GAATTC' at the EcoRI/NcoI fusion. Assuming that we might be dealing with a mix of DNA with (minority) and without the EcoRI site, BCCM/GeneCorner tried to purify the culture via single colony, selecting for the clear presence of the EcoRI site; without result however.
|History of deposit:||This plasmid was deposited by Dr V. Goossens(1)(2) and Dr J. Grooten(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:
|pCAGGSrhCAT (LMBP 3481) is available at BCCM/GeneCorner. This plasmid was deposited by Dr V. Goossens and Dr J. Grooten.|
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.