Gene insertion confirmation and copy-number quantification. (A) The five-gene insertion was confirmed by PCR with five pairs of gene specific internal primers. The PCR products revealed five specific bands: 810 bp (kan), 1012 bp (egIII), 1068 bp (cbhI), 750 bp (gfp), and 896 bp (NpaBGS). (B) The order of the gene cassettes was confirmed by PCR with five internal primer pairs. The PCR products resulted in six specific amplicons. (C) The relative ratios of the inserted gene copy numbers relative to the alg9 gene in KR5.