Figure 3.

Schemes of applied insert selections. For each selection type one vector example is shown. Positive selection with chloramphenicol: Vector with inactive promoter, upon insertion of target the -35 box is introduced and the promoter is active. Positive selection with kanamycin: By insertion of the target PCR a new C-terminal Phe residue is introduced which activates the kanamycin kinase. Wild Type kanamycin kinase has two Phe residues at the C-terminus. Positive selection with ampicillin: Similar to the kanamycin selection, the missing C-terminal Trp residue of beta-lactamase is complemented by the insert cloning. Negative selection with ccdB: The cassette with the cell killer gene ccdB is replaced by the desired insert.