Restriction digests of the clone give the following sizes (kb): HindIII--5.8, 1.7, 1.0; EcoRI--7.4, 1.1; BglII--8.5. The fission yeast tagging vectors, pSLF172 (ATCC 87609), pSLF272 (ATCC 87610) and pSLF372 (ATCC 87611), 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 C-terminus with triple HA tag, which contains an internal BamHI site. The vector does not contain an ATG, which must be provided by the insert. 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. |
