Amplification and Cloning of Membrane Protein Genes

Genes encoding the membrane proteins were amplified from genomic DNA using iProof High-Fidelity DNA Polymerase (Bio-Rad #172-5302). Francisella tularensis subsp. tularensis SCHU S421 (link) genomic DNA was provided by Drs. C. Rick Lyons, Terry H. Wu, and Jason Zsemlye (University of New Mexico). ASFV genomic DNA from the highly virulent isolates Georgia 2007/152 (link) and Malawi Lil 20/153 (link) was provided by Dr. Linda K. Dixon (The Pirbright Institute, United Kingdom). Unless noted, cloning was achieved using the In-Fusion HD Cloning Plus system (Clontech #638910). This ligation-independent system allows simple matching between 15 base pairs of each end of a PCR-derived insert with the ends of the linearized parent vector. Plasmid DNA was prepared with the QIAprep Spin Miniprep or Plasmid Maxi system (QIAGEN #27106 and #12163, respectively). DNA elutions were done using molecular grade water. Sequence confirmation of the complete insert in each plasmid clone was performed at the School of Life Sciences DNA Laboratory at Arizona State University. Vectors used in this study and representative primers are in Supplementary Tables 2 and 3, respectively.