TABLE 1.
Primers and sequencesa
| Primer | Sequence | Restriction enzyme |
|---|---|---|
| C5′ | 5′-AATACCATGGAAAATACAATACCCTTTAAT-3′ | NcoI |
| C3′ | 5′-TCTTCTGAATTCACTCTGGAAGATTATAGACAG-3′ | EcoRI |
| D5′ | 5′-GAAACCATGGTCCCTTTTAATATTTTTTCA-3′ | NcoI |
| FUP (D3′) | 5′-GTTTTCCCAGTCACGAC-3′ |
a
Incorporated restriction enzyme sites are underlined. PCRs contained 1× PCR buffer; dATP, dCTP, dGTP, and dTTP (200 μM each); 5′ and 3′ primers (0.5 μM each); DNA template (1 or 0.5 ng); MgCl2 (1.25 mM); and Taq DNA polymerase (2.5 U) (Applied Biosystems, Foster City, Calif.). Typical PCR protocol was as follows: initial heating to 94°C for 1 min and then cycles of 94°C, 45 s; 54°C, 30 s; and 72°C, 45 s, for a total of 30 cycles. Products were initially cloned into pGEM-T (Promega Corp., Madison, Wis.) prior to subcloning into ProEXHT-A (Life Technologies, Mt. Waverley, Australia) expression plasmid. All constructs were confirmed by DNA sequencing.