Construction of a broad-host-range kanamycin-resistant plasmid vector.

A new 10.2-kb plasmid, pIRL2, was constructed by using a 2140-bp DNA fragment carrying the Kmr gene and BamHI cohesive ends. These BamHI cohesive ends were used to trap the replicating DNA fragment from a partial Sau3A digest of the plasmid R300B. The plasmid contains unique EcoRI, SstI, HindIII, SmaI, SalI, and XhoI sites. These sites can be used as cloning sites without the loss of Kmr. A unique BglII site can be used as a cloning site by insertional inactivation of the Kmr structural gene, coding for neomycin phosphotransferase type II. The new plasmid carries the Sur and Smr genes of R300B. The direction of transcription from the neo promoter is clockwise, the same as that from the sul promoter. The plasmid retains the broad-host-range function of R300B, and thus it may be used for gene cloning in Rhizobium and Agrobacterium for genetic engineering of plant cells.