Abstract
Synthetic phage platforms are robust microbiology tools with therapeutic potential against antimicrobial-resistant bacteria. Here, we present a protocol for rebooting Pseudomonas phages with a terminally redundant, circularly permuted 65 kbp genome. We describe steps for designing PCR primers to generate DNA fragments, reconstituting the complete linear phage genome, performing seamless in vitro assembly, and finally, purifying and electroporating the DNA using a P. aeruginosa clinical isolate.