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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMRS101 (LMBP 3654)

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Price category: Cat. 3 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3654.gb (View with Genome Compiler)
p3654.txt
p3654.pdf
Sequence
analysis results
Genecorner:

NGS: c02-gc-dec2018.fasta

Cloned DNA: Bacillus subtilis levansucrase cDNA (sacB, GeneID 936413)
Escherichia coli insertion element IS1 protein InsB cDNA (insB)
Promoter: -
Ribosome
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
Gram-negative bacterial strains
Parental clone: pKNG101; pSK02
Further information: The plasmid was constructed by inserting the E. coli pMB1 ori and the ampicillin resistance gene as a NotI fragment of pSK02 into the unique NotI site of pKNG101. pSK02 is a derivative of pBluescriptIISK+, in which SspI-XbaI and BssHII-AflIII fragments were deleted by digesting, filling-in and religating. The XbaI site, created by ligating a filled-in XbaI to a SspI site, is destroyed.
pMRS101 is 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. It additionally contains 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 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. Streptomycin remains a selection marker for integration.
The plasmid contains two XbaI sites; the one located between the ampicillin resistance gene and the R6K origin 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 LT727367.1, except for the changes made after NGS sequencing, including the absence of the T3 promoter.
EMBL Accession number: LT727367.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 12/02/2020
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BamHI, BssHII, BssHII/EcoRI, EcoRI, NotI, SmaI and XbaI.

This plasmid has also been fully sequenced.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr G. Cornelis(1).
(1) Microbial Pathogenesis Unit, UCL-ICP, Brussels, Belgium
Plasmid reference: Sarker et al., Mol. Microbiol. 23 (1997), 410-411 [PMID: 9044275]
Related plasmid reference: Kaniga et al., Gene 109 (1991), 137-141 [PMID: 1756974]
Fettes et al., Int. J. Med. Microbiol. 290 (2000), 239-250 [PMID: 10959726] [DOI: 10.1016/S1438-4221(00)80121-6]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 LK111
Host reference: Zabeau et al., EMBO J. 1 (1982), 1217-1224 [PMID: 6327257]
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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