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For direct assembly of transformable plasmids by cycled ligation, a modified pUC19 vector (Table S1) with two blunt ligation sites separated by ∼250 bp was created. The vector was digested with NruI and SwaI (NEB) and the linearized vector was isolated and purified by gel extraction (Qiagen). Detailed protocol in Table S3. |
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Protocol tips |
For direct assembly of transformable plasmids by cycled ligation, a modified pUC19 vector (Table S1) with two blunt ligation sites separated by ∼250 bp was created. The vector was digested with NruI and SwaI (NEB) and the linearized vector was isolated and purified by gel extraction (Qiagen). Detailed protocol in Table S3. |
Publication protocol
Both PCR products were treated with SacI and Aor51HI, and simultaneously cloned into the SacI site of the pFA6a-hphMX6 vector to obtain pSTk14. Next, a DNA fragment containing the full-length of the trt1+ gene was amplified using the following primer set.
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