Reactions concerned with the one-tube protocol for generation of promoter reporter gene fusions. (A) To generate the GFP Destination Vector, the B0464.4::gfp Expression Clone was linearized by digestion in the promoter segment with SalI before use in a BP recombination. Transformation of DB3.1 cells allowed recovery of plasmids containing the ccdB cassette. Selection for ampicillin resistance yielded the desired plasmids. (B) To recover the promoter Entry Clone, pDONR P4P1R was linearized by digestion in the ccdB cassette with BamHI before use in a BP reaction. Transformation of DH5α cells selected against plasmids containing the ccdB cassette and with selection for kanamycin resistance only the desired plasmid was obtained. (C) In the one-tube protocol, promoter::reporter gene fusions would be generated by carrying out the BP and LR recombination steps successively, in a single tube. Bold arrows indicate steps involved and crosses indicate recombination reactions.