Extended Data Table 3. Plasmids used to generate yeast expression strains.
Synthetic constructs were codon optimised for expression in Saccharomyces cerevisiae34. Sequences from the 5′ and 3′ end of the PGK1 gene were also added. Genes were synthesised by GeneArt Gene Synthesis (Life technologies).
| Plasmid | Original vector* | Insert | Plasmid construction |
|---|---|---|---|
| pTD2 | pJF2 | Dpb11 | PCR product from S. cerevisiae W303 genomic DNA using primers Dpb11 fwd and Dpb11 reverse cloned 5′ - SgrAI , 3′ - NotI |
| pTD5 | pJF2 | Sld2 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pTD6a | pJF2 | Sld3 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pTD6b | pJF4 | Sld7 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS303-Cdc45 | pJF2 | Cdc45 | PCR product cloned from S. cerevisiae W303 genomic DNA using primers cdc45 fwd and cdc45 rev 5′ - SgrAI , 3′ - NotI |
| pRS303-Cdc45iFlag1 | pJF2 | Cdc45iFlag1 | Site directed mutagenesis using oligonucleotides JTY32 and JTY33 and pRS303-Cdc45 as template |
| pRS303-Cdc45iFlag2 | pJF2 | Cdc45iFlag2 | Site directed mutagenesis using oligonucleotides JTY40 and JTY41 and pRS303-Cdc45iFlag1 as template |
| pRS304 (Pol2 + Dpb4-CBP) | pJF18 | Pol2 Dpb4-CBP |
Pol2 - synthetic construct cloned 5′ - AscI , 3′ - XhoI Dpb4-CBP - synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS306 (Dpb2 + Dpb3) | pJF19 | Dpb2 Dpb3 |
Dpb2 - synthetic construct cloned 5′ - SgrAI , 3′ - NotI Dpb3 - synthetic construct cloned 5′ - AscI , 3′ - XhoI |
| pRS305/Gal-CBP-Top2 | pCG001 | Top2 | Synthetic construct cloned 5′ - AscI , 3′ - XhoI |
| pRS303/Gal-CBP-Ctf4 | pJF2 | Ctf4 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS303/Gal4 + CBP-Tev-Clb5 | pJF2 | Clb5 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS306/Cks1-Gal-Cdc28 | pJF5 | Cks1 Cdc28 |
Cks1 - synthetic construct cloned 5′ - SgrAI , 3′ - NotI Cdc28 - synthetic construct cloned 5 - AscI , 3 - XhoI |
| pRS303/Gal4 + CBP-Tev-Rfa1 | pJF2 | Rfa1 | Synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS306/Rfa2-Gal-Rfa3 | pJF5 | Rfa2 Rfa3 |
Rfa2 - synthetic construct cloned 5′ - AscI , 3′ - XhoI Rfa3 - synthetic construct cloned 5′ - SgrAI , 3′ - NotI |
| pRS304/Pol1-Gal1-10-Pol12 | pJF3 | Pol1 Pol12 |
Pol1 - synthetic construct cloned 5′ - SgrAI , 3′ - NotI Pol12 - synthetic construct cloned 5′ - AscI , 3′ - XhoI |
| pRS306/CBP-Tev-Pri1-Gal1- 10-genomic Pri2 | pJF5 | Pri1 Pri2 |
Pri 1 - synthetic construct cloned 5′ - AscI , 3′ - XhoI Pri2 - PCR product from S. cerevisiae W303 genomic DNA using primers JTY92 and JTY100 cloned 5′ - AscI , 3′ - XhoI |