Oligonucleotides primers used in this study
| Name | Sequence |
|---|---|
| clb2-0*† | 5′-GGATCCTATAGATGTCCAACCCAA-3′ |
| clb2-1* | 5′-CGCGGATCCGAATTCATGAGTGAAACTGATAGTGGA-3′ |
| clb2-2* | 5′-CGCGGATCCGAATTCATGGACCTAGATGCAGAAGATGT-3′ |
| clb2-3* | 5′-CGCGGATCCGAATTCATGAATATTCATCAAAATCGAGATATC-3′ |
| clb2-4* | 5′-CGCGGATCCTCAGCGGCCGCCATGCAAGGTCATTATATCAT-3′ |
| clb2-9† | 5′-GCGCTCGAGTTCGGTATTTCTAACTTGAAGTTGACT-3′ |
| clb2-10† | 5′-GCGGTCGACCTAGATGCAGAAGATGTAAATGATCCA-3′ |
| clb2-F/A‡ | 5″-CATCGGATTGGGATAATTTAGGTTAGCTTTCAATGTCTTTAAAATG-3′ |
| clb3-1§ | 5′-CGCAGATCTGAATTCAAAATGCATCATAACTCACAGTC-3′ |
| clb3-2§ | 5′-CGCAGATCTTCCGGGTTGGGTTTATACAGGTC-3′ |
| clb3-3§ | 5′-CGCGGATCCGGATAAACAACCAGAGTTAAGA-3′ |
| clb3-4§ | 5′-CGCGAATTCAGCGGCCGCTGTTAGATCTTTCTACTCTGTG-3′ |
| bud3-1¶ | 5′-TTACGTGTTCGGGCTCTTATCTGGTTGCTAAAAGAGTATATTTACACCTCACCACGGATCCCCGGGTTAATTAA-3′ |
| bud3-2¶ | 5′-AATGTATACATTGCATTAAATTAAAAAGAAAAAAAAAATCAATAAAACACGAATTCGAGCTCGTTTAAAC-3′ |
| bud3-3¶ | 5′-TGGGAAACAGAACTGCGCTGTTGGTGGCCCAGAGAAACTGAAATTTTATCGGATCCCCGGGTTAATTAA-3′ |
| Bud3-4** | 5′-GCGCCGCCAGCTTCAAAAATTAACTTTCAAAGGTCACCATCCTATATTCGGATCCCCGGGTTAATTAA-3 ′ |
| Bud3-5** | 5′ATAATGGGCATCATGCTTATCTAAAACCACCCTCATTCCTTGAAAGAGGAATTCGAGCTCGTTTAAAC-3 ′ |
↵* Primers used for PCR amplification of truncated mutants of CLB2 (Δ132, Δ176, Δ213, Δ255). Underlined nucleotides correspond to restriction sites used for cloning into the pMJ200 vector and pRS316GAL1-HA3.
↵† Primers used for the construction of the Δ176-213 internal deletion of CLB2. Underlined nucleotides correspond to the restriction sites used for the internal fusion (clb-9 and clb-10) and for cloning into the pMJ vectors and pRS316GAL1-HA3 (clb2-0 and clb2-14 or clb2-4).
↵‡ Primers used for F354/A in vitro mutagenesis. Underlined nucleotides correspond to introduced mutations.
↵§ Primers used for amplification of CLB3 full-length, C-ter and N-ter domains. Underlined are restriction sites for chimeras construction and cloning into pMJ300, and pRS316GAL1-HA3.
↵¶ Primers used for PCR-based BUD3 deletion (1 and 2) and CFP tagging (3 and 2).
↵** Primers used for PCR-based construction of the bud3Δ1221 allele.