TABLE 2.
Primers used to prepare plasmids expressing EGFP-M2 truncations
| Constructa | Primer sequence (5′ to 3′)b
|
|
|---|---|---|
| Forward | Reverse | |
| pEGFP-N-M2(1-41) | CGATTCCTCGAGGCAAAGATGGGGAACGC | CGATCGAAGCTTATTTAACATTCCAGGTG |
| pEGFP-C-M2(582-708) | ATACTCGAGGAATGGGTGTACGGATATTCAATC | AAGCTTTCAGCGTGTATACCCACGCTTAACC |
| pEGFP-C-M2(585-708) | ATCTCGAGGTATATTCAATCCCAAAGGAATTCTC | ATAAGCTTTCAGCGTGTATACCCACGCTTAACCAC |
| pEGFP-C-M2(610-675) | ATCTCGAGCAATTACGCAGGCAGCACCGGT | TATAAGCTTTCAAGGCGCTGGAAAGTGCGTCGA |
| pEGFP-C-M2(582-611) | ATACTCGAGGAATGGGTGTACGGATATTCAATC | TAAAGCTTTCAAATAATTGTACTTGGATCACC |
| pEGFP-N-M2(582-611) | ATACTCGAGGAATGGGTGTACGGATATTCAATC | TAAAGCTTAATAATTGTACTTGGATCACC |
| pEGFP-C-M2(582-643) | ATACTCGAGGAATGGGTGTACGGATATTCAATC | ATAAGCTTCACCCCGCCGATAAACTTTTAGTACG |
| pEGFP-C-M2(582-675) | ATACTCGAGGAATGGGTGTACGGATATTCAATC | TATAAGCTTTCAAGGCGCTGGAAAGTGCGTCGA |
| pEGFP-C-M2(582-675ΔH2)c | (i) ACCCCAACGAGAAGCGCGATCAC | (ii) ACCCCCGCCGATAAACTTTTAGTAATAATTGTACTTGGATCACC |
| (iii) TGATCCAAGTACAATTATTACTAAAAGTTTATCGGCGGGGGTG | (iv) TGGACAAACCACAACTAGAATGCAGTG | |
Each construct was designed to express an EGFP fusion including the indicated amino acid residues of μ1. The pEGFP-C1 and -N1 vectors and all PCR products were digested with XhoI and HindIII for cloning.
Forward primers are in the same orientation as the coding strand; reverse primers are in the reverse orientation to the coding strand. Restriction enzyme sites are in italics, and the inserted start and stop codons are underlined.
Primer pairs i and ii amplified a region in pEGFP-C-M2(582-675) from within EGFP to M2 residue 611. Additional sequence in reverse primer ii (bold type) was complementary to the 5′ sequence of M2(637-675). Primer pairs iii and iv amplified a region in pEGFP-C-M2(582-675) from M2 residue 637 to a region in the vector downstream of the M2 gene. Additional sequence in forward primer iii (bold type) was complementary to sequence at the 3′ end of M2(582-611).