Last data update: 28 November 2020 04:24 CET
Plasmid name: pMRS101-R (LMBP 4732)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Bacillus subtilis levansucrase cDNA (sacB, GeneID 936413)
Escherichia coli insertion sequence IS1 insB
|Promoter:||Phage T3 promoter|
|Selection marker:||Ampicillin (amp)
|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
|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.|
|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
|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)|
|Related host reference:||Simon et al., Biotechnology 1 (1983), 784-791
|Cultivation medium:||LB-Lennox + streptomycin (25 μg/ml)|
|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.