For the subfamilies arcA, bacA, and nuc with strong variation, we constructed two variants, "DM selective" and "DM general", with different parameter settings.
For that we combined information from CYP2D6 reference sequence M33388 (http://www.ncbi.nlm.nih.gov/nuccore/181303) and CYP2D6 variant database we constructed in the previous step.
To examine whether the promoter sequence variants influence transcription efficiency, we constructed luciferase reporter plasmids carrying TLR4 promoter sequences with single or multiple changes.
Thus, we constructed variants of pGG eC using different ent-CPP synthases, specifically that from corn (Zea maize, An2 (Harris et al. 2005)), from rice (Oryza sativa, OsCPS2 (Prisic et al. 2004)), and the bacteria Bradyrhizobium japonicum (BjCPS; Morrone et al. 2009).
Based on our previous findings about the absence of the Trp intermediate in four of the Synechocystis catalase-peroxidase variants on the heme distal side (W122F, W106A, H123Q, and R119A) we constructed new variants on Trp122 and Trp106 positions.
We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains.
To determine if the catalytic activity of the AAA+ and/or STK domains of YeaG are required for function, we constructed mutant variants of YeaG based on amino acid sequence alignments with AAA+ and STK domains of well-characterized proteins (Figure S1B and Figure S1C).
We constructed two variants, ptsNH73A and ptsNH73A/H120A, and tested them for their ability to suppress the requirement of σE for viability in the cotransduction assay.
To determine the relative affinity of G12 for this epitope, and for similar sequences present elsewhere in toxic prolamins, we constructed hexapeptide variants of the G12 epitope, two of which were designed based on their presence in the prolamins of barley and rye (Figure 4A and 4C).
To further improve the AGO2/shRNA system, we constructed several variants of shEGFP417 with different loop sizes (Fig. 3c).
