TABLE 1.
NEBuilder primers used for detection of T407A and S411A Kunjin viruses
| Fragmenta | NEBuilder primerb | Primer sequenceb,c |
|---|---|---|
| Fragment 1 | CMV forward | atcggaatctGATTATTGACTAGTTATTAATAGTAATCAATTACG |
| CMV reverse | gcgaactactCGGTTCACTAAACGAGCTC | |
| Fragment 2 | ||
| 5′ T407A virus | 5′ Kunjin forward | tagtgaaccgAGTAGTTCGCCTGTGTGAG |
| Kunjin virus | 5′ Kunjin (T407A) reverse | atatatctgtGGCGACGACAAAGTCCCAATC |
| Fragment 3 | ||
| 3′ T407A virus | 3′ Kunjin (T407A) forward | tgtcgtcgccACAGATATATCTGAGATGGG |
| Kunjin virus | 3′ Kunjin reverse | gtcaataatcTTCCGATAGAGAATCGAG |
| Fragment 2 | ||
| 5′ S411A virus | 5′ Kunjin forward | tagtgaaccgAGTAGTTCGCCTGTGTGAG |
| Kunjin virus | 5′ Kunjin (S411A) reverse | ctcccatctcTGCTATATCTGTTGTGACGAC |
| Fragment 3 | ||
| 3′ S411A virus | 3′ Kunjin (S411A) forward | agatatagcaGAGATGGGAGCAAACTTTAAG |
| Kunjin virus | 3′ Kunjin reverse | gtcaataatcTTCCGATAGAGAATCGAG |
a
The mutant Kunjin viruses were generated from three fragments, fragments 1, 2, and 3. The product of fragment 2 from the NEBuilder assembly reaction contains the specified mutation.
b
The underlined primers for fragments 2 and 3 contain the alanine mutation at either position 407 or position 411 (underlined).
c
The sequences indicate the 5′ overlap sequence (lowercase nucleotides)/spacer sequence/and the sequence annealed at the 3′ end (uppercase nucleotides).