Fig. 2

Preparation of key parts.(a) Preparation of CEN3, using K. phaffii from gDNA. Left-hand gel: PCR amplicons of segment (Seg) 1 (oligos 197/198), and Seg 2 (oligos 199/200). This approach introduces one extra, functionally silent, base pair (G:C) in the core region (see Additional file 1: Fig. S2). DNA bands of expected sizes (boxed) were extracted and digested with appropriate restriction enzymes before sequential cloning into pUC19 to yield eDA24. Right-hand gel: restriction digestion of eDA24 confirming presence of 6.3-kb CEN3 (IR = inverted repeats). (b) Assembly of framework plasmid in a pUC19-derived scaffold (ori = bacterial origin of replication) (eDA53 validation shown in Additional file 1: Fig. S3). (c) The oligos used to construct the telomeres part (Tel, Additional file 1: Fig. S4). Each proto-telomere contains 16 copies of TGGATGC and the two are linked by the 18-bp I-SceI-recognition site. Lower schematic: cloning of Tel into eDA40 (eDA131 validation shown in Additional file 1: Fig. S5)