Figure 1.
Expression vector design and assembly. (a) The standard expression vector contains a medium-strength constitutive promoter, RBS1, which allows for strong translation initiation of a leader peptide, and a translationally coupled, variable RBS2, mediating translation initiation of the coding sequence (CDS) of the membrane protein of interest.18 (b) Vectors are assembled with different BCD elements. First, the vector is amplified by PCR, subsequently it is digested by type IIS restriction enzymes. The latter allows for seamless assembly with a library of annealed oligo pairs (encoding the different BCD variants), which have overhangs complementary to the digested vector.