Multi-component assembly of a chimaeric scaffoldin - ‘Scaf4L’. (a) Schematic representation of the four components that were amplified in the first PCR reaction: A. cellulolyticus cohesin (A) and C-terminal linker segment, B. cellulosolvens cohesin (B) and linker, carbohydrate binding module (CBM) (c) and linker and C. thermocellum cohesin (T) but without the C-terminal linker. (b) Resultant PCR products. (c) The PCR products were then used as mega-primers for the restriction-free reaction with the pET28a plasmid. The sequences that form base-pairing due to flanking regions designed in the primers of each module are indicated by the coloring of the PCR products and by connecting lines. (d) The resulting linear plasmid pET28a-scaf4L. Ligation occurs spontaneously in E. coli.