10 beta competent escherichia coli

Zolpidem was purchased from Toronto Research Chemicals. GABA, kanamycin, theophylline, collagenase, HEPES, and all salts or other chemicals not specifically mentioned were purchased from Sigma-Aldrich and were of analytical grade. Oligonucleotides were purchased from Sigma-Aldrich, and sequencing services were from Australian Genome Research Facility. Restriction enzymes, Q5 polymerase, T4 DNA ligase, and 10-beta competent Escherichia coli were from New England Biolabs. DNA purification kits were from Qiagen. The QuickChange II Site-Directed Mutagenesis kit was from Agilent Technologies, and the mMessage mMachine T7 transcription kit was from ThermoFisher Scientific.

All plasmids and oligonucleotides used in this study are listed in Supplementary Table S1 and Supplementary Table S2, respectively. For cloning, two 30 bp overlapping oligonucleotides were designed and amplified using Q5^® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions. The resulting PCR product was purified (QIAquick PCR Purification Kit, Qiagen), digested with AgeI-HF and NheI-HF (NEB) and ligated into equally digested pWHE601* with T4 DNA Ligase (NEB).
Doped pools were generated using the oligonucleotides AgeI_doped_fwd and NheI_[3.0/4.5/9.0/30.0]_doped_rev (Supplementary Table S3, Microsynth AG), respectively, with the construct ΔATG as template and amplified using Q5^® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions. Again, digestion and ligation into pWHE601* followed. Transformation of the ligation mixture was done after butanol precipitation into NEB^® 10-beta Competent Escherichia coli (High Efficiency) according to the supplier's protocol. This ensured that the number of different plasmids yielded by this process were >50,000.