Fig. 2. Assembly strategy for the generation of artificial myxochromide BGCs consisting of four steps: ligation of synthetic mch gene fragments on the cloning vector pSynbio1; ‘desplitting’ of gene constructs using type IIS restriction enzymes; ‘rejoining’ of the single domain encoding fragments after removal of splitter elements (SEs); reconstitution of the BGC by stepwise assembly of the promoter, 5′-/3′-truncated gene, intergenic linker and terminator fragments on the expression vector pSynbio2. Restriction sites (R) were introduced in SEs as well as at both termini of each gene synthesis fragment (RL, KpnI; RR, PmeI; RT, PvuI; other R-sites are listed in Table S3†). RIIS, type IIS R-site. Recognition sequences for IIS endonucleases used in the SEs (BsaI or AarI) were introduced in the flanking regions of mchA′/B′/C′ gene fragments for the desplitting process (Table S3†).
