Fig. 6

The COOH-terminus tagging plasmid. a Schematic representation of pNX3a-HA3 and sequence of three tandem HA and multi cloning sites. The HA encoding gene is inserted between PacI AscI sites. The 100 base Tag primer anneals to the left end (pink arrowhead on the left in plasmid image and long arrow in HA3 sequence) and Bot primer anneals to the right end to amplify indicated cassettes (pink arrowheads on the right in plasmid image). A number of unique restriction enzyme sites are indicated. b Sequence of three tandem PK (top) and FLAG (bottom) fragments. pNX3a-PK3 and pNX3a-FL3 plasmids were generated by replacing HA3 sequence between NheI and XbaI sites in pNX3a-HA3 (a) with indicated PK and FLAG sequences, respectively. c Detection efficiency of PK-tagged Tpz1. Western blot shows detection of PK epitope fused Tpz1. No obvious non-specific bands were detected. Proteins were extracted from cells and subjected to SDS-PAGE. Anti-V5 antibody (AbD Serotec) was used to detect PK fused Tpz1 protein. Anti-Cdc2 antibody (anti-PSTAIRE) (Santa Cruz) was used as a control for loading. d Telomere length homeostasis is slightly impaired with the nine tandem PK tagging of Tpz1. Genomic DNA was harvested from cells cultured over 2 weeks after generation of strains, and digested with EcoRI and separated in 1 % agarose gel. Telomere containing fragments were detected with the synthetic telomeric DNA probe