Pool oligonucleotides (Integrated DNA Technologies) were as follows:
5´–[P]–CGAGNNNNNNNNNNNNNNNNNNNNNNNNNC–3´
5´–[P]–ACGAGNNNNNNNNNNNNNNNNNNNNNNNNN–3´
…Wherein 5´–[P] denotes a 5´ Phosphate, and N denotes an equimolar mixture of all four nucleotides. Oligonucleotides were each resuspended in annealing buffer (10 mM Tris, pH 7.0, 50 mM NaCl) to 100 µM. 10 µL of each oligo were mixed in a 0.2 mL PCR tube; this mixture was heated to 95°C for 10 minutes and slowly annealed to 25°C over the course of two hours in a thermocycler. The reaction was snap-cooled on ice and diluted 100-fold with ice-cold annealing buffer. 1 µL of this diluted duplex mix was ligated into 25 ng of BbsI-cut sgINTgpc, in 12 µL final volume, using the Quick Ligation Kit (New England Biolabs). The entire reaction was transformed into 120 µL of XL10-Gold ultracompetent cells (Agilent), plated onto 12 LB Ampicillin plates and grown overnight at 37°C. Seven bacterial colonies were picked for Sanger sequencing (Supplementary Fig. 9 ), and the remainder were pooled by scraping the plates into 100 mL of liquid LB(Amp). Bacteria were pelleted by ultracentrifugation, and the plasmid pool was harvested in a single plasmid maxi-prep (QIAgen) (Supplementary Figs. 9–10 ).
5´–[P]–ACGAGNNNNNNNNNNNNNNNNNNNNNNNNN–3´