Restriction digests of the clone give the following sizes (kb): BamHI--3.5, 1.7; EcoRI--2.7, 2.3; PstI--1.9, 1.4, 0.95, 0.9; PvuII--4.0, 1.1; HindIII/SmaI--5.2. Transfected cells amplify DHFR sequences. Encodes the L22F mutant of DHFR that can serve as a dominant selection marker because it retains catalytic activity and also confers methotrexate (MTX) resistance at low MTX concentrations (125 nM). Constructed by cloning a HindIII/SmaI fragment containing the gene into pSV2-neo from which neoR had been removed. This plasmid was then cut with SmaI and BglII to remove 18 G nucleotides at the 3' end. The BglII site was filled in, and ligated to the SmaI site, thereby destroying the original BglII and SmaI sites. Removing the HindIII/SmaI fragment from pSV2-neo removes all but the 3' 171 bp of neoR. |
