Extended Data Fig. 3: Library construction and characterization pipeline.

a) Library construction procedure. Step 1) Clone a codon-optimised R. rubrum rubisco sequence into pUC19. Step 2a) Choose locations to split the gene which are appropriate for the cloning of subpool libraries. Step 2b) PCR amplify the sub-libraries from an oligo pool containing all 8778 mutations. Step 3) PCR amplify the backbone with a space missing for the ligation of an oligo subpool. Step 4) Ligate each oligo subpool to its appropriate backbone. Step 5) Combine the sub libraries, cut the full, mutated genes out and ligate them into a PCR-amplified and barcoded backbone. After transformation scrape the desired number of colonies for selection. b) Library sequencing strategy. The library was characterised by long read sequencing. Barcode abundances were measured by short-read sequencing before and after selection (see methods).