TABLE 2.
Primers used for amplification of targeted genesa
| Gene | Accession no. | Directionb | Sequence (5′–3′)c | Location | Expected size (bp) |
|---|---|---|---|---|---|
| p17 | AF330703 | F | CGGAATTCATGCAATGGCTCCGCCATACGA (EcoRI) | 293–314 | 441 |
| R | GCTCTAGATCATAGATCGGCGTCAAATCGC (XbaI) | 733–712 | |||
| p171-118 | AF330703 | F | CGGAATTCACAATGCAATGGCTCCGCCATACG (EcoRI) | 293–313 | 354 |
| R | AAACTCGAGTCAGGATTGAGACCCGCCATCCCAATG (XhoI) | 646–623 | |||
| PRAS40 | NM_032375.5 | F | TAGAATTCAAGATGGCGTCGGGGCGC (EcoRI) | 793–807 | 782 |
| R | CGGTCGACCTCCCTGGACTTCAATATTTCC (SalI) | 1574–1553 | |||
| PRAS40T246A | NM_032375.5 | F | TAGAATTCAAGATGGCGTCGGGGCGC (EcoRI) | 793–807 | 782 |
| R | TAGTCGACTCAATATTTCCGCTTCAGCTTCTGGAAGTCGCTGGCGTTAAGCCG (SalI) | 1563–1519 | |||
| Rheb | NM_005614.4 | F | CAGAATTCAAGATGCCGCAGTCCAAGTC (EcoRI) | 382–401 | 558 |
| R | TTGTCGACTCACATCACCGAGCATGAAG (SalI) | 939–920 |
a
Primers used for creating pcDNA3.1-p17, pcDNA3.1-p-171-118, pCI-neo-PRAS40, pCI-neo-PRAS40 T246A, and pCI-neo-Rheb vectors for transfection.
b
F, forward; R, reverse.
c
Underlines indicate restriction enzyme digestion sites in the primers.