Figure 1

Principle of parallel SLIC cloning with negative ccdB selection. The vector is PCR linearized with LP1 forward and LP2 reverse primer. The LP1 primer corresponds to PreScission protease site (3C) for tag removal. The LP2 primer is either located at the C-terminus of ccdB or corresponds to a C-terminal tag. In both cases the ccdB gene is deleted upon PCR amplification thereby allowing counterselection of parental empty vector in ccdB sensitive cells. The Gene of Interest (GOI) is PCR amplified with primers composed of 5’ and 3’ gene specific sequences plus 15 bp – 25 bp extensions complementary to LP1 and LP2 vector primers, respectively.