NruI NEB#R0192

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Upstream tips	Protocol tips	Downstream 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.

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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