The pIS-0 vector (plasmid 12178; Addgene, Cambridge, MA, United States) (Yekta et al., 2004) (see Supplementary Figure 3) was linearized with SacI-HF® and BmtI-HF® restriction endonucleases (New England Biolabs, Ipswich, MA, United States) and purified using QIAquick® PCR spin columns (Qiagen, Germantown, MD, United States). Plasmid assembly was performed using 40 ng of linearized plasmid and 2 μL of unpurified PCR product containing double-stranded test oligonucleotides using the NEBuilder® HiFi DNA assembly kit (NEB, Ipswich, MA, United States) per manufacturer’s instructions. The universal primers used to amplify the test-oligonucleotide pool also served as the flanking homology regions for the NEBuilder® assembly. Two μL of the NEBuilder® assembly product were transformed into 60 μL chemically competent E. coli (transformation efficiency > 5 × 108 cfu/μg) (Takara, Mountain View, CA, United States) and plated on six standard 100 mm LB-agar plates containing 100 μg/ml ampicillin. After overnight incubation, all colonies were dislodged from the plates by adding 2 ml LB-broth containing 100 μg/ml ampicillin and agitation using 10–20 ColiRollersTM glass beads (EMD Millipore, Billerica, MA, United States). The colonies harvested from the
