Fig. 3

synIII design and synthesis. a synIII design. Twenty-one retrotransposons (RT) and seven introns were removed. Forty-three TAG stop codons were changed to TAA stop codons. Ninety-eight loxPsym sites were introduced to enable SCRaMbLE analysis. The two natural telomeres were replaced with shorter universal telomere caps. A single copy of essential tRNA gene SUP61, which codes for tRNASer (CGA), was deleted and moved to a tRNA neochromosome. Numerous PCR-Tags were incorporated into synIII to distinguish it from the natural counterpart. As a result, synIII is about 13.8 % smaller than the native yeast chromosome III (Box 2). For the complete set of additions, deletions and other genome modifications to synIII, see Annaluru et al. [32]. b synIII synthesis. synIII was constructed in three steps (shown in the flow diagram on the left, from bottom to top). In step 1, 750 bp building blocks (BB) were synthesized from 60-mer oligonucleotides at Johns Hopkins University by undergraduate students in the Build-A-Genome course [33]. In step 2, three to five BB were assembled into 2–4 kb minichunks by homologous recombination in Saccharomyces cerevisiae [35]. Adjacent minichunks were designed to encode overlap of one BB to facilitate downstream assembly. In step 3, direct replacement of native yeast chromosome III with pools of synthetic minichunks was performed. Eleven iterative one-step assemblies and replacements of native genomic segments of yeast chromosome III were carried out using pools of overlapping synthetic DNA minichunks, encoding alternating genetic markers (LEU2 or URA3), which enabled complete replacement of native III with synIII in yeast [32]. The number of oligonucleotides, BBs, and minichunks needed to construct synIII are shown in parentheses. SynIII is 272,871 bp long, compared with the 316,667 bp long native yeast chromosome III