Generation and Cloning of MS2 Variant Libraries

Single-stranded DNA primers were purchased that spanned the length of each 26-codon region of MS2 with appropriate cut sites for each corresponding entry vector (Supplementary Data 4). Primers for each entry vector were resuspended, pooled, and diluted to a final concentration of 50 ng/µL. The reverse strand was filled in using a touchdown PCR^{62 (link)} with 10-mers directed to the golden gate cut sites. The amplified, double-stranded DNA was purified using a PCR Clean-up Kit (Promega, Cat# A9282), then diluted to 1–5 ng/µL. These mixtures were cloned into their respective entry vectors using EMPIRIC cloning^{48 (link)}, which relies on established golden gate cloning techniques^{60 (link)}. The ligated plasmids were transformed into chemically competent DH10B E. coli and plated on large (245 × 245 × 20 mm, #7200134, Fisher) LB-A plates with 32 µg/mL chloramphenicol. Colony number varied, but every transformation yielded a number of colonies that was at least three times the theoretical library size. This protocol was repeated in full for three total biological replicates that are fully independent from library generation through selection.