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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMKSV40/8-4 (LMBP 652)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p652.gb (View with Genome Compiler)
p652.txt
p652.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Simian virus 40 (SV40); Gluzman mutant
Promoter: Simian virus 40 early promoter (SV40 early)
Simian virus 40 late promoter (SV40 late)
Ribosome
binding site:
-
Terminator: Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Kanamycin (kan)
Replicon: Escherichia coli plasmid ColE1 origin
Host range: Escherichia coli
Parental clone: pMK16
Further information: The plasmid was constructed as follows: 1) first, the pMK16 plasmid was modified by eliminating the BglI site via a deletion (20% of total genome) in the region of the tetracycline resistance gene (cleavage with BglI and treatment with S1 nuclease); 2) the complete nucleotide sequence of SV40 was then inserted as a BamHI fragment into the unique BamHI site of the BglI(R) pMK16 derivative; 3) the SV40 sequence was then mutated by deletion of 4 nucleotides of the BglI site of the ori region, destroying the origin of viral DNA replication.
The plasmid is useful for constructing cell lines that constitutively produce large T- and small t-antigen (cfr. COS cells).
The SV40 late promoter is presumably inactive, due to the mutation in the SV40 ori (Contreras et al. (1982)).
Name mentioned in Gluzman et al. (1980) is 8-4. Name mentioned in Gheysen et al. (1983) is pMK16/8-4.
EMBL Accession number: -
Latest sequence update: 10/10/1994
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BamHI, BglI, BspTI, Eco47III and KpnI. The restriction site pattern does not completely fit the nucleotide sequence file that we compiled based on the publication of Gluzman et al., Cold Spring Harbor Symposia on Quantitative Biology 44 (1980): it seems that, in the area between or around the Eco47III sites at positions 6017 and 6732 (between the SV40 VP1 sequence and the kanamycin resistance gene), a fragment of approximately 700 bp is not present.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr W. Fiers(1) (2), who received it from Dr Y. Gluzman(3).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
(3) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
Plasmid reference: Gluzman et al., Cold Spring Harb. Symp. Quant. Biol. 44 (1980), 293-300 [PMID: 6253143]
Related plasmid reference: Kahn et al., Methods Enzymol. 68 (1979), 268-280 [PMID: 232215]
Contreras et al., Nature 300 (1982), 500-505 [PMID: 6292733]
Gluzman et al., Proc. Natl. Acad. Sci. U.S.A. 77 (1980), 3898-3902 [PMID: 6254000]
Gheysen et al., J. Virol. 47 (1983), 1-14 [PMID: 6306266]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 DH1(λ)
Host reference: Bachmann, in 'Escherichia coli and Salmonella: Cellular and Molecular Biology', American Society for Microbiology, Washington DC (1987), 1190-1219
Cultivation medium: LB-Lennox + kanamycin (50 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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