We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6Kγ DNA replication origin (oriRR6Kγ) so they replicate only in bacteria supplying the Π replication protein (encoded bypir), and they can be maintained at low or high plasmid copy number by usingEscherichia colistrains encoding either wild-type or mutant forms of Π. They also carry the RP4 transfer origin (oriTRP4) so they can be transferred by conjugation to a broad range of bacteria. Most of them encodelacZα for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carriestetAR(from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carriessacB(fromBacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pirhost (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetSor SucRrecombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.