Fig 2.
Construction and features of pRL128. (A) Different cloning steps during construction of the pRL128 plasmid template. The parental pKD13 vector is a template plasmid used in the λ red technology (38). A Ptac-lacO DNA fragment was amplified with primers carrying 5′ extremities corresponding to DNA sequences of pKD13 symbolized by rectangles 1 and 2. This Ptac-lacO fragment was inserted into pKD13 by RF cloning (see Materials and Methods). Then, an araC-PBAD DNA fragment was inserted into pRL127. For pRL128, P1 and P2 arrows indicate the orientation and location of the primers carrying the priming site sequence P1 and P2 (see Table S2 in the supplemental material). (B) Sequences of the pRL128 regions around the Ptac and PBAD promoters. Promoter sequences are underlined. The lacO sequence is indicated in bold letters. Priming sites 1 and 2 are indicated by arrows.