Fig. 4

Shuffling of residue M203 of hChR2-mCherry by degenerate primers and by the modified Gibson assembly method. a Depiction of the hChR2-mCherry synthetic gene (colored segments, A and B). Fragment A includes sequences spanning from the standard promoter of the plasmid and intracellular linker III (ILIII; purple); fragment B spans from ILIII to the end of the mCherry (orange). Backbone of plasmid is light grey. The degenerate sequence (green) is situated between purple and orange fragments. Sizes (# of bps) of the fragments are noted within the fragments. Intrinsic and unique digestion sites are noted (BamHI and BsrGI). Primers used for amplification of each fragment are noted on the right (with corresponding colors). b Image of PCR products from step I on 1% agarose gel. DNA ladder sizes (in Kbp) are noted on the right of ladder. c Left lane shows the products of the Gibson assembly prior amplification. Bottom bands show non-assembled fragments. The expected ‘assembled’ product is not detectable (dashed box, ~ 2 Kbp), whereas the amplified assembled Gibson product by CAG promoter_F and the FP_R primers is easily noticeable (right lane; arrowhead). d Digested vector by BamHI and BsrGI. e Sequences, and matching chromatograms, of DNA sequences obtained from resulting colonies (labeled with dashed box)