Restriction digests of the clone give the following sizes (kb): BamHI--8.5; EcoRI--7.3, 1.2; PstI--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 1) amplification by PCR with primers designed to flank the triple HA tag in Bluescript-HA and to modify the polylinker, 2) gel purification of the PCR product and digest with XhoI and 3) ligation into REP4X cleaved with XhoI and SmaI. |
