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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pMSK1-E-D565A (LMBP 4982)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4982.gb (View with Genome Compiler)
p4982.txt
p4982.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human mitogen- and stress-activated protein kinase-1 cDNA (MSK1, RPS6KA5); kinase-dead mutant
E-tag; C-terminal
Promoter: Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Phage T7 gene 10 promoter (T7g10)
Phage SP6 promoter
Ribosome
binding site:
-
Terminator: Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Suppressor tRNA gene (supF; requires a p3 containing host)
Replicon: Bacteriophage M13 origin
Escherichia coli plasmid pMB1 origin
Polyoma virus origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli; use strains with the p3 helper plasmid (kan resistant, amber codon in amp and tet)
Mammalian cells; SV40 permissive cells
Parental clone: pMSK1-E
Further information: The plasmid was derived from pMSK1-E by PCR-based site-specific mutagenesis, creating a kinase-dead mutant of the human MSK1 cDNA. As compared to the wt human MSK1 cDNA, the aspartic acid (D) codon at position 565 was replaced by an alanine (A) codon.
pMSK1-E-D565A is a shuttle vector designed for stable or high-level transient expression of mutated human MSK1 in mammalian cells. The inserted gene is expressed under control of the hCMV-IE promoter and enhancer and the SV40 polyadenylation site.
A small HIV (human immunodeficiency virus) fragment (29 nucleotides) between the hCMV-IE and T7 promoter sequences has an enhancer function.
The T7 and SP6 promoters can be used for in vitro transcription of the cDNA insert.
The presence of the polyoma and SV40 origins of replication enables the vector to replicate episomally in mammalian cells expressing large T-antigen (e.g. WOB, COS cells).
The supF tRNA suppressor gene is used for selection in E. coli MC1061[p3]. It is recommended to select on both ampicillin and tetracycline containing media.
The M13 origin is useful for synthesis of single-stranded DNA upon infection with the M13 helper phage.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727062.1.
The nucleotide sequence of the wt human MSK1 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF074393.1. The region of the mutation was sequenced by BCCM/GeneCorner.
Other names of the plasmid are pcDNA1-hMSK1(D565A) and pcdnamskI_Asp565Ala.
EMBL Accession number: AF074393.1, view at EMBL, GenBank, DDBJ
LT727062.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 06/02/2006
Sequence detail:
560                 565
wild type hMSK1: 5' ... GAA.ATT.AAA.ATA.ATT.GAT.TTT ... 3'
                        Glu Ile Lys Ile Ile Asp Phe
  
                        560                 565
mutant hMSK1:    5' ... GAA.ATT.AAA.ATA.ATT.GCT.TTT ... 3'
                        Glu Ile Lys Ile Ile Ala Phe
                                             ^
                                   
^: mutated nucleotide.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BsrDI, HindIII and NotI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr L. Vermeulen(1) and Prof. Dr G. Haegeman(1). It was constructed by Dr G. De Wilde(1).
(1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Vermeulen et al., EMBO J. 22 (2003), 1313-1324 [PMID: 12628924]
Related plasmid reference: Reber et al., PLoS ONE 4 (2009), e4393 [PMID: 19197368] [DOI: 10.1371/journal.pone.0004393]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061[p3]
Host reference: -
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Invitrogen Instruction Manual
Helper plasmid: p3
Cultivation medium: LB-Lennox + ampicillin (50 μg/ml) + kanamycin (50 μg/ml) + tetracycline (10 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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