Table 1 Primers designed and used for PCR amplification and cloning of zymogen (no leader) Ae. aegypti mosquito midgut proteases. Primers were purchased from ELIM Biopharmaceuticals, Inc. The melting temperature (TM) of the primer sequence that anneals to the gene of interest was estimated using NetPrimer (Premier Biosoft, Palo Alto, CA). The restriction enzyme used for each primer are underlined and in bold
Protease | Primer | Primer Sequence | TM (°C) |
|---|---|---|---|
AaET | AaET-Zym-pET-Fwd | 5’-AAAAACATATGGCAACGCTGTCCAGCGGTC-3’ | 64.55 |
AaET | AaET-Zym-pET-Rev | 5’-AAAAAAAGCTTATTAAACCTCGGAAACCTCTCGGA-3’ | 64.24 |
AaSPVI | AaSPVI-No Leader-Fwd | 5′ –AAAAACATATGGCTTCAACCGGTGGTTTGC– 3’ | 61.1 |
AaSPVI | AaSPVI-Zym-pET-Rev | 5′ –AAAAAAAGCTTATTACAATCCACTGACCTCCTGG– 3’ | 59.09 |
AaSPVII | AaSPVII-Zym-pET-Fwd | 5’-AAAAACATATGCTATCAACCGGATTCCATCCGC-3’ | 65.36 |
AaSPVII | AaSPVII-Zym-pET-Rev | 5’-AAAAAAAGCTTATTAAACTCCACTGACTTCCGCCA-3’ | 63.72 |
AaLT | AaLT-Zym-pET-Fwd | 5’-AAAAACATATGTTCCCATCGTTGGACAACG-3’ | 59.59 |
AaLT | AaLT-Zym-pET-Rev | 5’-AAAAACTCGAGTTATTACAGTCCAGTCTTCTGCTTGA-3’ | 57.11 |