Last data update: 18 January 2021 04:24 CET
Plasmid name: pCAGGS-TSE (LMBP 4174)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
Trypanosoma cruzi trans-sialidase gene (TS); mature sequence|
Phage fd gene 3 (g3); fragment
|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-TS; pMa58; pCAhIFNGSTmE|
|Further information:||The plasmid was constructed as follows: 1) The Trypanosoma cruzi trans-sialidase coding sequence (TS) was isolated as a StuI/MscI fragment from pCAGGS-TS and ligated into the SmaI opened pMa58 vector, resulting in the intermediate construct pMa58TSvoll; 2) A unique StuI site was introduced in front of the termination codon of the mature TS gene by site-specific mutagenesis using the synthetic linker 5'-CCCCTGATCAGGCCTTCGTGTCGCTGCTGC-3'; 3) Finally, the mature TS gene was isolated as a BssHII (filled in with Klenow DNA polymerase)/StuI fragment and ligated into the XhoI/MunI (filled in with Klenow DNA polymerase) opened pCAhIFNGSTmE vector, leading to pCAGGS-TSE.
pCAGGS-TSE is useful for highly efficient expression of trans-sialidase, C-terminally fused to an E-tag, 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.
Other name of the plasmid is pCAGGS-TSjE.
|EMBL Accession number:||-|
|Latest sequence update:||25/08/2000|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BglII, HincII, HindIII/XbaI and StuI.|
|History of deposit:||This plasmid was deposited by Prof. Dr N. Callewaert(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Laroy et al., Protein Expr. Purif. 20 (2000), 389-393 [PMID: 11087678]
|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-TSE (LMBP 4174) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr N. Callewaert and was published in Laroy et al., 2000.|