Fig 1. The recombineering protocol adapted for multiple rounds.

First, the dP kinase activity of hsvTK is used for rapid and efficient counter-selection in liquid media. The mechanism of action of dP-selection is shown in the left panel. Second, the number of clones to be transferred between the steps is limited by diluting/aliquoting the transformant cultures, to prevent the propagation and subsequent domination of clones with PCR-generated inactivating mutations. Finally, the gene coding for hsvtk was duplicated to drastically reduce the emergence of selection escapes. TMK: thymidylate kinase, NDK: nucleoside diphosphate kinase, DNA pols: DNA polymerases, Km: kanamycin.