a, Recoding landscape of fragment 9. Our designed synthetic sequence of fragment 9 was integrated into the genome by REXER, and 19 clones were completely sequenced by next-generation sequencing. The recoding landscape graph shows the frequency at which each target codon was recoded across the 19 clones. Although most codon replacements were accepted, recoding of a 26-kb region was consistently rejected; codon positions with a recoding frequency of zero in all the sequenced clones are indicated by black dots. To pinpoint the problematic sequence, 10-kb stretches of the genome (labelled G2 to G7) were deleted in the presence of the episomal copy of synthetic fragment 9. The synthetic sequence was sufficient to support deletion of all stretches except G4 (dark grey box), which suggests that an underlying problem is within this stretch. None of the nineteen clones was completely recoded. b, Recoding landscape of stretch G4. After REXER across the 10-kb G4 stretch, and sequencing of 10 clones, the recoding landscape shown was generated. This revealed a clear recoding minimum at yceQ—a ‘gene’ that encodes a predicted protein for which there is little evidence of transcription, protein synthesis or homologues37. All target codons in yceQ were recoded at least once in individual clones, but never simultaneously; thus, the minimum of the recoding landscape does not reach zero, and 0/10 clones were completely recoded. This is consistent with epistasis between the targeted positions. In the map below the recoding landscape, sequences annotated as essential are shown in dark grey and target codons are shown in red. The sequence position (x axis) is with reference to a. c, Altered design of the region surrounding rne in fragment 9. Top, original design of yceQ recoding and rne (which encodes RNase E) regulatory sequences. Target codons are shown in red. P1rne, P2rne and P3rne are the promoters (blue arrows) for the essential gene rne; these are found in and around the hypothetical gene yceQ. The −10 sequence of the major promoter P1rne is mutated by our initial design. The sequences that contains hairpin 1 (hp1) and hairpin 2 (hp2), which bind to RNase E to mediate transcript degradation, are shown as blue bars; these sequences encompass the remaining target codons and are also mutated by our initial design. Bottom, the second codon in yceQ was replaced with a stop codon (purple) and the remaining target codons retained their original sequence. The sequence position (x axis) is with reference to a. d, The modified fragment 9 (from c) was integrated on the genome, which resulted in complete recoding in 4/5 clones that we sequenced. The axes of the graph are the same as in a. The recoding landscape for the modified fragment 9, derived from sequencing five clones, is shown in purple. The data from a are reproduced for comparison.