Fig. 2.

General concept of assembly of designed nucleotides (26); two strategies for the linking of fragments are possible (cases I and II). In case I the two genes, A and B, that are to be virtually shuffled are aligned, the different colored stars refer to information that encodes different amino acids, whereas oligonucleotide fragments with both colored stars in the same position of the parent gene denote the synthetic oligonucleotide fragment with degenerate nucleotides. The gray blocks denote conserved regions of sequence that can be used as the linking part with homologous recombination. Case II shows no homology between flanking oligos, which can be assembled by ligation between single-stranded DNA with unknown terminal sequences.