Last data update: 04 December 2020 04:17 CET
Plasmid name: pLOF/Km (LMBP 3337)
|Price category:||Cat. 3 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Escherichia coli lac repressor gene (lacI)
Escherichia coli IS10R transposase gene
|Promoter:||Escherichia coli hybrid tryptophan/lacUV5 promoter (tac)
Escherichia coli lac repressor promoter; mutant (lacI(q))
|Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)|
|Selection marker:||Ampicillin (amp)
Neomycin (neo; kanamycin (kan))
|Replicon:||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 construction of the plasmid is described in Herrero et al. (1990).
pLOF/Km is a Tn10 based transposon vector delivery plasmid.
The transposition system consists of the IS10R transposase gene located outside the mini-Tn10 element and driven by the tac promoter, and MluI-SfiI-NotI-MluI sites between the inverted repeats of the Tn10 transposable element.
The SfiI site can be used for the cloning of foreign DNA fragments with the help of the specialized cloning vector pUC18Sfi.
The plasmid carries the kanamycin resistance gene, inserted as a NotI fragment; constructs lacking an insert between the inverted repeats do not seem to be viable.
The plasmid is unable to replicate in E. coli host strains devoid of the R6K-specified õ protein of the pir gene.
The plasmid contains the RP4 conjugal transfer origin (oriT) for transfer to a variety of Gram-negative bacteria.
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 E. coli strains expressing the tra genes of RK2/RP4 (e.g. S17-1(λpir)).
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727467.1.
The nucleotide sequence of the E. coli kanamycin-resistant Tn10-based transposon cassette corresponds with the EMBL Nucleotide Sequence Database accession number AJ601386.1.
|EMBL Accession number:||AJ601386.1, view at EMBL, GenBank, DDBJ
LT727467.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||09/10/2008|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BamHI/Bsp68I, BglII/EcoRI, EcoRV/MluI, NotI/SphI, PstI/SacI and SfiI.
This plasmid has also been fully sequenced but the NGS sequence data still need to be implemented.
|History of deposit:||This plasmid was deposited by Dr K.N. Timmis(1). It was constructed by Dr M. Herrero(1) and Dr V. de Lorenzo(1).
(1) GBF, Braunschweig, Germany
|Plasmid reference:||Herrero et al., J. Bacteriol. 172 (1990), 6557-6567 [PMID: 2172216]
|Related plasmid reference:||De Lorenzo and Timmis, Methods Enzymol. 235 (1994), 386-405 [PMID: 8057911]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 S17-1(λpir)|
|Host reference:||Tascon et al., J. Bacteriol. 175 (1993), 5717-5722 [PMID: 8396122]
|Related host reference:||Simon et al., Biotechnology 1 (1983), 784-791
Kalogeraki et al., Gene 188 (1997), 69-75 [PMID: 9099861]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml) + kanamycin (50 μg/ml)|
|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.