Last data update: 20 October 2020 04:14 CEST
Plasmid name: pCAMFhGNTIf1 (LMBP 3124)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|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
|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.|
|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)|
|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.