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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPNEcad (LMBP 2828)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p2828.gb (View with Genome Compiler)
p2828.txt
p2828.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse cadherin 1 cDNA (Cdh1, E-cad, CDH1)
Promoter: Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter
Phage T3 promoter
Ribosome
binding site:
-
Terminator: Mouse phosphoglycerate kinase 1 polyadenylation signal (PGK1 polyA)
Selection marker: Ampicillin (amp)
Neomycin (neo; G418)
Replicon: Escherichia coli plasmid ColE1 origin
Host range: Escherichia coli; preferably recombination-deficient strains
Mammalian cells
Parental clone: pPNT; pBATEM2
Further information: The plasmid was constructed as follows: 1) First, an intermediate construct was made by inserting the small (filled-in) NcoI-XmaI fragment from pBATEM2 between the Eco47III and XmaI sites of pPNT. This inserted fragment contains part of the E-cadherin gene and a small 3' UTR fragment. 2) The E-cadherin sequence was then retrieved by replacing the NheI-HindII fragment of the intermediate vector with the NheI-Ecl136II fragment from pBATEM2.
pPNEcad contains the complete coding sequence of the mouse E-cadherin gene with 33 nucleotides of the 5' UTR and 34 nucleotides of the 3' UTR.
The Tn5 neomycin resistance gene contains some modifications at the start of the coding sequence as compared to the wild type gene.
As the plasmid contains the mouse PGK1 promoter and polyA sequence twice, one should be aware of the possibility of deletions due to homologous recombination.
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 LT727548.1.
Name mentioned in Keirsebilck et al. (1998) is pPNECad.
Other names of the plasmid are pPNECAD-N and pPNECAD-N(NheI).
EMBL Accession number: LT727548.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 21/11/1996
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: AgeI, ApaI, EcoRI, EcoRV, EcoRV/XhoI and XhoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by A. Keirsebilck(1) (2), K. Staes(1) (2) and Prof. Dr F. Van Roy(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Keirsebilck et al., Invasion Metastasis 18 (1998), 44-56 [PMID: 10207250]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 DH5α
Host reference: Focus 8 (1986), 9
Related host reference: Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

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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