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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMc519 (LMBP 2026)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p2026.gb (View with Genome Compiler)
p2026.txt
p2026.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Escherichia coli alkaline phosphatase A gene (phoA); signal sequence
Promoter: Escherichia coli hybrid tryptophan/lacUV5 promoter (tac)
Ribosome
binding site:
Ribosome binding site (RBS) of the Escherichia coli alkaline phosphatase A gene (phoA)
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)
Terminator: Phage fd terminator
Selection marker: Chloramphenicol (cam)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Parental clone: pMc58
Further information: The plasmid was designed for site-specific mutagenesis where the mutated sequence can be physically linked with a selectable marker (amp).
The E. coli phoA signal sequence starts with ATG, while in the wild-type sequence the start codon is GTG.
Adventitious coding sequences can be joined in phase to the phoA signal sequence using the blunted KpnI site.
Only 13 nucleotides upstream the ATG start codon of phoA are derived from the original phoA ribosome binding site.
The plasmid provides resistance to chloramphenicol, but is sensitive to ampicillin in sup(-) strains.
More than 1 mutagenesis round is possible via switching from chloramphenicol resistance to ampicillin resistance.
pMc519 contains the replication origin of the single-stranded DNA phage f1, so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage, e.g. M13KO7.
When cloning a fragment downstream from the tac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
After mutagenesis, use mutS strains for primary transformation (e.g. sup(-) strain WK6mutS, sup(+) strain BMH71-18mutS); for segregation of possible mutants: sup(-) strains (e.g. WK6).
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727618.1.
EMBL Accession number: LT727618.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 09/08/1990
Sequence detail:
Nucleotide sequence at the last codon of the phoA signal sequence:

                 *
5' GTG.ACA.AAA.GCG.GTA.CCC.GGG.GAT.CCT.CTA.GAG.TCG.ACC 3'
                 KpnI  SmaI          XbaI    SalI 

*: end of the signal sequence of phoA.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BanII, EcoRI and HincII.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr P. Stanssens(1).
(1) Corvas International N.V., Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Stanssens et al., Nucleic Acids Res. 17 (1989), 4441-4454 [PMID: 2501754]
Gibson et al., Biochemistry 45 (2006), 6363-6371 [PMID: 16700547] [DOI: 10.1021/bi060288q]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 WK6
Host reference: Zell et al., EMBO J. 6 (1987), 1809-1815 [PMID: 3038536]
Cultivation medium: LB-Lennox + chloramphenicol (25 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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