Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions and the absence of methods that enable the stepwise replacement of the E. coli genome with long synthetic DNA, and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress on this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (~100 kb) synthetic DNA, through the in vivo excision of double stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda red mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.The design and synthesis of genomes provides a powerful approach for understanding and engineering biology1-6. Genome synthesis has the potential to elucidate synonymous codon function7, accelerate metabolic engineering8, and facilitate genetically encoded unnatural polymer synthesis9,10.Methods that i) replace the genome in sections6, ii) provide feedback on precisely where a given design fails and on how to repair it, and that iii) can be rapidly iterated for whole

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