Gibson isothermal assembly

All molecular biology was performed following standard procedures (Maniatis et al. 2012 ) and either Gibson Isothermal Assembly (Gibson et al. 2009 (link)) or Gateway Cloning (Invitrogen, Carlsbad, CA). One Shot Mach1 T1 Phage-Resistant Chemically Competent Escherichia coli (Life Technologies) or Mix & Go Competent Cells - Strain Zymo 5α (Zymo Research, Irvine, CA) were used for chemical transformations. DNA was prepared using QIAprep Spin Miniprep or Midiprep Kits (QIAGEN, Valencia, CA) and QIAquick Gel Extraction, and PCR Purification Kits (QIAGEN) or Zymoclean Gel DNA Recovery Kit (Zymo Research) were used to extract or purify DNA fragments, respectively. All restriction enzymes were purchased from New England Biolabs (NEB) (Beverly, MA). Gibson Isothermal reaction mix was either purchased (NEB) or created from a published recipe (Gibson et al. 2009 (link)). All plasmids were sequence-verified and sequencing services were provided by GATC (GATC Biotech AG, Konstanz, Germany), Source Biosciences (Nottingham, UK) or Macrogen (Rockville, MD). Oligonucleotide synthesis was carried out by Integrated DNA Technologies (Coralville, IA) and gene synthesis by Epoch Life Science (Sugar Land, TX).