Table 2.
Plasmids used in this study
| Plasmid | Source |
|---|---|
| pAC24 | URA3 ADE3 plasmid (2μ) (23) |
| pAC39 | SacI–BamHI genomic Cdc23p fragment (4.7 kb) cloned into pAC24 (URA3 ADE3, 2μ) |
| pAC173 | Original YCp50 isolate encoding Sac3p (URA3 CEN) |
| pAC174 | Genomic PstI–PstI fragment (5.8 kb) of pAC173 encoding Sac3p cloned into pRS315 (LEU2 CEN) |
| pAC175 | pAC174 was digested with XhoI, and oligonucleotides encoding the myc epitope were inserted (LEU2 CEN) |
| pAC45 | Nuf2p-GFP fusion plasmid with a unique XhoI site at the ATG of sGFP (S65T, V163A), used for making C-terminal GFP fusion proteins (24) |
| pAC60 | SAC3 was amplified by PCR by using an oligo 5′ of the SAC3 stop codon with a SalI site. The PCR product was cut with SnaBI (329 bp upstream of SAC3 ATG) and SalI and ligated into pAC45 cut with SmaI and XhoI to create SAC3-GFP (URA3, 2μ). |
| pAC176 | pAC60 cut with XhoI and ApaI to liberate a 1.8-kb fragment encoding the C-terminal 321 bp of Sac3p fused to GFP. This fragment was cloned into pAC174 cut with XhoI and ApaI (SAC3-GFP LEU2 CEN). |
| pAC61 | pAC60 was cut with HpaI and StuI to liberate the Sac3p-GFP fusion lacking the first 96 bp of SAC3 (including the ATG). This fragment was cloned into pRS303 (25) to generate a truncated integrating Sac3-GFP construct (HIS3). |
| pRS202 | URA3 plasmid backbone (2μ) for the high-copy library (gift from P. Hieter, Univ. of British Columbia) used to screen for suppressors |
| pAC17 (pPS311) | LEU2 CEN plasmid with a GAL1-10 promoter followed by several unique sites including BamHI and SalI |
| pAC188 | Oligonucleotides with a 5′ BamHI and a 3′ SalI site were used to amplify the SAC3 gene. The PCR product was cloned into pAC17 (LEU2 CEN GAL1-10) |
| PAC342 | Nup49p fused in-frame to GFP (26) |
| pAC403 (pPS892) | PGEX.2TB inserted into pPS293 (GAL1-10 URA3 CEN) (23) |
| PAC404 | Oligonucleotides with a 5′ BamHI and a 3′ SalI site were used to amplify the SAC3 gene. The PCR product was cloned into pAC403 to create an N-terminal GST fusion. |
| pAC485 | Oligonucleotides with a 5′ BamHI and a 3′ SalI site were used to amplify the NTF2 gene. The PCR product was cloned into pAC403 to create an N-terminal GST fusion. |
| pAC361 | Oligonucleotides with a 5′ BamHI and a 3′ SalI site were used to amplify the PRP20 gene. The PCR product was cloned into pAC403 to create an N-terminal GST fusion. |
| pAC213 | NES-NLS-containing GFP reporter protein (7, 10) |
| PAC520 pLDB419 | YAP1∷GFP LEU2 2μ plasmid with sGFP inserted at the NdeI site of YAP1 gene (21) |