Mutagenic libraries were generated on Ace-mNeon-T2A-NLS-mCherry and VARNAM-T2A-NLS-mCerulean backbones using a pre-established protocol (18 (link)). Briefly, a set of 4 forward primers, containing degenerate codons WKC, NMC, VWG, or DGG at the target site, was pooled with a single, partially overlapping reverse primer, and mutagenizing PCR reactions were set up using CloneAmp™ polymerase (Clontech). Following DpnI treatment to digest unmutated template, the linear PCR products were circularized using InFusion® ligase (Clontech) and transformed in TOP10 competent cells (Invitrogen). To obtain up to 19 unique AA substitutions at a single target site, 48 colonies were picked and cultured in 96 deep-well culture plates. Plasmid DNA was isolated using Nucleospin® 96 Plasmid kit (Macherey-Nagel) on an epMotion 5075 liquid handling workstation (Eppendorf), and purified DNA was collected in 96-well plates. The libraries were sequenced after voltage screening to identify the individual mutations and ensure at least 19 variants were obtained at every site.