Figure 2.

Generation of ‘long’ homology arm negative-selection recombineering replacement cassettes via In-Fusion cloning. (A) PCR products, generated from template bMON14272 and primer pairs 6054–6055 and 6059–6060, containing 3′ and 5′ gp64 flanking sequences were fused in an Overlap-PCR with primer pair 6054–6060 to produce a single PCR product with central, unique SnaBI site and terminal SmaI sites that was cloned into pGEM-T Easy to create pMW009. (B) RT-cassette and replacement cassettes, containing coding sequences for SeMNPV F, AdhoNPV F and AcMNPV gp64 genes, were generated by PCR, with respective primer pairs 6005a–6006a, 6009a–6010a, 6063–6064 and 6106–6107, and cloned, via In-Fusion cloning, into pMW009 generating, respectively, pMW012, pMW013, pMW033 and pMW024.