Last data update: 24 January 2024 16:39 CET
Plasmid name: pCAGGS-TSE (LMBP 4174)
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| 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.