Restriction digests of the clone give the following sizes (kb): BamHI--5.8, 4.4, 3.2; BglII--6.6, 5.2, 1.6; HindIII--13.5. The portion of the construct encoding PLAT consists of a genomic BclI/BglII fragment (approximately 2.3 kb) beginning in the intron 5' to the initiation AUG, and extending through an intron into the region encoding the mature polypeptide. The rest of the PLAT sequences are derived from a 1.97 kb BglII cDNA fragment encoding the complete processed polypeptide. PLAT expression is regulated by the LTR, the mutant dhfr by an SV40 early promoter, and G418 resistance by a thymidine kinase promoter. The dhfr-IV mutation decreases enzyme affinity for methotrexate and permits resistance to higher levels of the drug. The order of the major features in this plasmid is: SV40 polyadenylation - <- dhfr-IV - SV40 early promoter - HaSV LTR - ClaI - PLAT - SV40 polyadenylation - tk promoter - neoR - SV40 polyadenylation. |
