Construction of gene replacement vectors for Gram− bacteria using a genetically modified sacRB gene as a positve selection marker – Springer

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JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser. Based on the wide-host-range suicide vector pSUP102 (Simon et al. 1983), gene replacement vectors (pWS232/pWS233) have been developed that facilitate the identification by a positive selection procedure of double recombination events in Gram bacteria. The vectors contain the tetracycline and gentamicin resistance gene as selectable markers, as well as a modified phenotype. Although the Tn5 insertion was flanked on one side by only approximately 100 bp of VF39 homologous DNA, this was sufficient for homologous recombination to occur, and double recombinants could readily be isolated. Arnold W, Pühler A (1988) A family of high copy number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179 Gay P, Le Coq D, Steinmetz M, Berkelmann T, Kado CI (1985) Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J Bacteriol 164:918–921 Kaniga K, Delor I, Cornelis GR (1991) A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106 cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis. Gene 57:239–246 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467 Simon R, Hötte B, Klauke B, Kosier B (1991) Isolation and characterization of insertion sequence elements from Gram-negative bacteria by using new broad-host-range, positive selection vectors. J Bacteriol 173:1502–1508 Wohlleben W, Arnold W, Bissonnette L, Pelletier A, Tanguay A, Roy PH, Gamboa GC, Barry GF, Aubert E, Davis J, Kagan SA (1989) On the evolution of Tn Source.

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