Last data update: 24 January 2024 16:39 CET
Plasmid name: pCAMFhGNTIf1 (LMBP 3124)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3124.gb
(View with Genome Compiler) p3124.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF) Human N-acetylglucosaminyltransferase I cDNA (GlcNAc-TI, MGAT1); mature sequence |
| 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; pSCGAL1MF3; pUChGNTI1 |
| Further information: | This plasmid was constructed as follows: i) the XbaI-StuI fragment of pSCGAL1MF3, containing the prepro secretion signal sequence (ppMF) of the Saccharomyces cerevisiae α-mating factor 1 gene (MFα1), was ligated to the XbaI-Eco47III vector fragment of pUChGNTI1; ii) this intermediate construction was then digested with HindIII and EcoRI, the sticky ends were filled in with Klenow DNA polymerase and EcoRI linkers (5'-CGGAATTCCG-3') were added; iii) finally, this EcoRI-EcoRI fragment was digested with XbaI and EcoRI and the resulting XbaI-EcoRI fragment was inserted into the XbaI-EcoRI digested pCAGGS vector. This expression plasmid carries the ppMF sequence of the S. cerevisiae MFα1 gene fused to the mature sequence of the human gene encoding N-acetylglucosaminyltransferase I, cloned in the sense orientation relative to the chicken β-actin/rabbit β-globin hybrid promoter (AG). 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 LT727444.1. The nucleotide sequence of the human GlcNAc-TI cDNA corresponds with the Genbank accession number M55621.1. |
| EMBL Accession number: | M55621.1, view at EMBL, GenBank, DDBJ LT727444.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 06/09/2002 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: ScaI, SpeI and XmnI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr R. Contreras(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Maras et al., Eur. J. Biochem. 249 (1997), 701-707 [PMID: 9395316] |
| Related plasmid reference: | Kumar et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 9948-9952 [PMID: 1702225] |
| 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 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pCAMFhGNTIf1 (LMBP 3124) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Contreras and was published in Maras et al., 1997. |
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.