LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMRS101-R (LMBP 4732)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: (View with Genome Compiler)
analysis results


Cloned DNA: Bacillus subtilis levansucrase cDNA (sacB, GeneID 936413)
Escherichia coli insertion sequence IS1 insB
Promoter: Phage T3 promoter
binding site:
Terminator: -
Selection marker: Ampicillin (amp)
Streptomycin (Sm)
Replicon: Escherichia coli plasmid pMB1 origin
Escherichia coli plasmid R6K origin; defective pir gene
Broad-host-range plasmid RK2/RP4 origin of transfer (oriT)
Host range: Escherichia coli; use strains supplying the R6K pir function
Gram-negative bacterial strains
Parental clone: pMRS101
Further information: The plasmid was derived from pMRS101 by reversing the NotI fragment containing the T3 promoter, the E. coli pMB1 ori and the ampicillin resistance gene. This resulted in a higher transformation efficiency for the host strain E. coli DH5α.
The strA and strB genes of RSF1010 encode streptomycin phosphotransferase as a selection marker for integration. The last 10 codons of the original strB coding sequence (NotI-EcoRV) are missing due to the construction. Streptomycin remains a selection marker for integration.
pMRS101-R is, like pMRS101, an improved version of the pKNG101 mobilizable suicide vector that facilitates the positive selection of double recombination events in Gram-negative bacteria, combining the pir(-) origin of replication of the plasmid R6K and the sucrose-inducible sacB gene of Bacillus subtilis encoding levansucrase as a positive selection marker for excision. The plasmid contains also the RK2/RP4 origin of transfer and an easily removable high-copy-number origin of replication (NotI fragment), derived from pMB1, which facilitates the isolation of large amounts of high-quality plasmid DNA.
The plasmid contains two XbaI sites, the one located between the T3 promoter and the R6K ori only cuts in dam-negative strains.
Introduce the plasmid by electroporation, or through conjugation, using plasmid RK2/RP4 transfer functions (provided in trans by RK2/RP4, RK2/RP4-derived vectors, other replicons carrying RK2/RP4 transfer functions (e.g. pRK2073) or strains expressing the tra genes of RK2/RP4).
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727178.1.
EMBL Accession number: LT727178.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 14/08/2003
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BssHII, BssHII/XbaI, NotI, SacII, SalI and XbaI. The BssHII site at position 4011 could not be experimentally confirmed.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr J.G. Leahy(1). It was constructed by P. Batchelor(1).
(1) Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, USA
Plasmid reference: -
Related plasmid reference: Sarker et al., Mol. Microbiol. 23 (1997), 410-411 [PMID: 9044275]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 SM10(λpir)
Host reference: -
Related host reference: Simon et al., Biotechnology 1 (1983), 784-791
Cultivation medium: LB-Lennox + streptomycin (25 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: Use only streptomycin to select for the host/plasmid combination, as the plasmid can be stressed by using ampicillin.
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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