Unless specified, all cloning reagents were from NEB and plasmid backbones were from Addgene (https://www.addgene.org/). Primers were synthesized by Thermo Fisher Scientific. All fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB, no. E2621X). All assemblies were transformed into either DH5α Extreme Efficiency Competent Cells (Allele Biotechnology, no. ABP-CE-CC02050) or Stbl3 Chemically Competent E. coli (Thermo Fisher Scientific, no. C737303). Positive clones were confirmed by restriction digest and Sanger sequencing (GENEWIZ).
