TABLE 2.
Primers used in this study
| Primer | Sequence (5′→3′)a |
|---|---|
| Gene cloning | |
| LYS20_gateway_Fw | GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACTGCTGCTAAACCAAATCC |
| LYS20_gateway_Rv | GGGGACCACTTTGTACAAGAAAGCTGGGTGTTAGGCGGATGGCTTAGTCCGC |
| Gene cloning and disruption of LYS20 allele | |
| LYS20_BamHI_Fw | ACTGGATCCGTATACTGCGTGCGCTTGAGATTC |
| LYS20_SacI_Rv | CTGGAGCTCGGACGAACTTTGCGCGAAGTGG |
| Site-directed mutagenesis | |
| lys20_S385F_Fw | CGATGATGTTGACTT*TATCATCAAGAACTTCCACGCAGAG |
| lys20_S385F_Rv | CTCTGCGTGGAAGTTCTTGATGATAA*AGTCAACAT |
| lys20_S385E_Fw | ATCGATGATGTTGACG*A*A*ATCATCAAGAACTTC |
| lys20_S385E_Rv | GAAGTTCTTGATGATT*T*C*GTCAACATCATCGAT |
| lys20_S385L_Fw | ATCGATGATGTTGACT*T*A*ATCATCAAGAACTTC |
| lys20_S385L_Rv | GAAGTTCTTGATGATT*A*A*GTCAACATCATCGAT |
a
The underlined nucleotides indicate BamHI and SacI restriction sites. The asterisks indicate the positions of the nucleotide mutation.