GREAT AT SMALL THINGS

0

GeneCorner plasmid details

Last data update: 25 October 2020 04:11 CET

Plasmid name: pCAGGS-TSE (LMBP 4174)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p4174.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Trypanosoma cruzi trans-sialidase gene (TS); mature sequence
E-tag; C-terminal
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
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-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.
Class: Recombinant plasmid
Type: Plasmid
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)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
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.

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.

New search