Fig. 1

Overview of the GoldenBac strategy. In the first step, single targets are PCR-amplified using primers with extensions homologous to the primers used for PCR-linearization of the selected vector and the purified PCR products are combined into single expression construct via a RecA-mediated Sequence and Ligation Independent Cloning or In-Fusion cloning strategy. Small tags for purification or detection can be easily included on the primer extensions and internal BsaI recognition sites can be removed in this step by “fragmentation” of the gene. In the second step, expression cassettes are released from the single expression constructs upon cleavage with the BsaI restriction enzyme (recognition sites shown in red) and simultaneously ligated into the destination vector, based on the inter-compatible overhangs flanking the cassettes. Single or multiple positions can be encompassed by short sequences flanked by BsaI sites with fitting overhangs, called dummies. Selection against kanamycin, present only on the destination vector, will result in recovery of the final co-expression construct with high efficiency, both due to negative background selection of the empty destination vector with the ccdB spacer and due to the enrichment of final product by self-removal of the BsaI site