Restriction enzymes and t4 dna ligase

PCR primers are listed in Supplementary Table S1. Primers were designed for the native and codon optimized MAP genes (MAP2121c, MAP3733c, MAP2121synth, and MAP3733synth) based on either MAP strain K-10 sequence data available from the NCBI database (NC_002944) or using the sequence from GenScript synthesized genes. All conventional PCR reactions were carried out using high fidelity Velocity DNA polymerase Kit (Bioline) in accordance with the manufacturer's instructions. Restriction enzymes and T4 DNA ligase were purchased from Roche Diagnostics (Mannheim, Germany) and New England Biolabs (Beverly, MA, USA) and used as per manufacturer's recommendations. Ligation reaction mixtures were purified using the High Pure PCR product purification kit (Roche).

Oligonucleotides were from Invitrogen or IDT and are listed in Supplementary Table S2. All strains and plasmids were confirmed by polymerase chain reaction (PCR) and DNA sequencing was performed at the Allan Wilson Centre, New Zealand. Plasmid DNA was prepared using Zyppy Plasmid Miniprep Kits (Zymo Research). DNA from PCR and agarose gels was purified using the GE Healthcare Illustra GFX PCR DNA and Gel Band Purification Kit. Restriction enzymes and T4 DNA ligase were from Roche or NEB.