| Description |
pT2F2-SAhygpA-NP21-Rev (Accession numbrer: AB609717, 8,156bp) is a Tol2 transposon-based promoter trap vector that selects for expressed genes by hygromycin-resistance. This vector was generated by reversing the orientation of the FRT-franked region of pT2F2-SAhygpA-NP21. The neo and pu-delta-tk genes are arranged in tail-to-tail orientation, which confers G418-resistance and puromycin-sensitivity. Recombination between inversely-oriented lox2272 flips the orientation of neo-pu-delta-tk, resulting in loss of G418-resistance and acquisition of puromycin-reistance. This feature allows for selection of homozygous mutant cells by G418/puromycin double resistance because two copies of neo-pu-delta-tk cassettes exist in homozygous cells. FRT sequences internally located to the Tol2-terminal inverted repeats (TIRs) are used to excise internal mutagenic sequences upon Flp expression, generating revertant cells to verify cause-and-effect relationship between mutation and phenotype.
SA, adenovirus splice acceptor; hyg, hygromcin phosphotransferase; pA1, mouse phosphoglycerate kinase 1 (Pgk1) polyA signal; P, mouse Pgk1 promoter; neo, neomycin phosphotransferase; pu-delta-tk, fusion gene between puromycin N-acetyltransferase and a truncated version of herpes simplex virus type 1 thymidine kinase; pA2, bovine growth hormone polyA signal; R150, 150-bp TIR sequence at the right end of Tol2; L200, 200-bp TIR sequence at the left end of Tol2. |
