Restriction digests of the clone give the following sizes (kb): HindIII--5.6, 1.7, 1.0; EcoRI--7.4, 1.2; BamHI--8.5. The fission yeast tagging vectors, pSLF173 (ATCC 87612), pSLF273 (ATCC 87613) and pSLF373 (ATCC 87614), contain three versions of the nmt1 promoter: full strength (nmt1), medium strength (nmt1*) and low strength (nmt1**), respectively. The weaker promoters (nmt1* and nmt1**) contain mutations that attenuate both repressed and induced levels of expression. Each version of the nmt1 promoter can be expressed at low or high levels in thiamine-free media. The vector was designed to tag expressed protein at N-terminus with triple HA tag, which contains an internal BamHI site. The vector lack a stop codon. The vector was constructed by cutting pSLF173 with SacI and XhoI, isolating the fragment containing the HA polylinker, and ligating into XhoI-SacI digested REP82X. |
