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GeneCorner plasmid details

Last data update: 20 October 2020 04:14 CEST

Plasmid name: pCAMFhGNTIf1 (LMBP 3124)

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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.

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