Plasmid #3 serves as a template for tagging target proteins with N-terminal CL7 and already contains the HIV Nef protein – inserted at HindIII/XhoI restriction sites – as a model CL7-tagged protein.
Transcription is driven by the T7 RNA polymerase, and the plasmid is designed for expression in E. Coli.
The CL7 tag for purification is fused to the N-terminus of the target protein, upstream of the SUMO cleavage site.
Plasmid #3 includes two protease sites: a SUMO and Sortase A cleavage site exist between the CL7 binding site and downstream target protein.
The plasmid includes an N-terminal thioredoxin (Trx) tag upstream of the CL7 tag.