Fig. 5

Genome integration efficiency of Golden Gate vectors without repetitive homologous sequences (a) and expression of eGFP obtained after integration into different genomic loci (b). Genome integration efficiency (fraction of positive clones) are shown for P. pastoris transformed with different Golden Gate vectors containing P _GAP -eGFP_ScCYC1tt: control plasmid (single eGFP), eGFP in position 2 with a repetitive transcription unit (TU1) in position 1 and 3 (‘loop-out’ control) and 4 quadruple combinations with eGFP in position 1, 2, 3 and 5 next to four other transcription units TU1–4) (a). Genome integration was verified by analyzing eGFP expression of each 16 (controls) and 22 clones (quadruple combinations), respectively. TU1–4 were cloned with the promoters and transcription terminators P _POR1 /RPS3tt, P _PDC1 /IDP1tt, P _ADH2 /RPL2Att and P _MDH3 /TDH3tt, respectively. Coding sequences of TU1–4 were derived from various non-essential intracellular-protein coding genes of P. pastoris with a length of 500–2000 bp. The influence of the genomic integration locus was analyzed using vectors containing only one transcription unit (P _GAP _eGFP_ScCYC1tt) (b). In both cases clones were screened under glucose surplus