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. 2016 Nov 7;113(47):13390–13395. doi: 10.1073/pnas.1613825113

Fig. 1.

Fig. 1.

Construction of strain SacCdc45, encoding chromosomally expressed affinity-tagged Cdc45. (A) Schematic of the DNA sequence insertion in SacCdc45. A DNA sequence encoding a c-myc and hexahistine affinity tag was integrated into the 3′ end of gene Saci_0177, encoding Cdc45, using a “pop-in, pop-out” approach. (B) PCR amplification across the 3′ end of Saci_0177 using a genomic DNA template generated 188- and 245-bp products in SacTK and SacCdc45, respectively, indicating successful insertion of the affinity-tag encoding sequence. (C) SacCdc45 expresses C-terminal affinity-tagged Cdc45. Western blot analysis was performed using whole-cell extracts from exponential cultures of the parental strain, SacTK, and SacCdc45, using an anti-His-tag antibody, and anti-TBP antibody as a control.