TABLE 1.
MRFV genomic loci targeted for viability testing and VIGS constructs generated
| Construct (same ID as insert) | PDS size/total insert size (nt) | Insert position in MRFV genome | Clone ID | Construct details (bp: fragment size; nt: fragment position in MRFV genome; pmol: fragment amount used in 10 µl Gibson reaction) | ||||
|---|---|---|---|---|---|---|---|---|
| SmaI/StuI‐cut pJL89 (4675bp) | Fragment 1 | Fragment 2 | Fragment 3 | Fragment 4 | ||||
| a1 | 120/183 | nt 127/128 (after the start codon of polyprotein) | pSM199‐223 | 0.06 pmol | 127 bp (nt 1–127) 0.1 pmol | 183 bp (a1 insert) 0.1 pmol | 3,105 bp (nt 128–3232) 0.05 pmol | 3,105 bp (nt 3233‐6337) 0.05 pmol |
| b1 | 120/201 | nt 3709/3710 (HEL/POL junction) | pSM163‐177 | 0.04 pmol | 3,709 bp (nt 1–3709) 0.05 pmol | 201 bp (b1 insert) 0.1 pmol | 2,628 bp (nt 3710–6337) 0.05 pmol | – |
| b2 | 231/312 | nt 3709/3710 (HEL/POL junction) | pSM326‐351 | 0.04 pmol | 3,709 bp (nt 1–3709) 0.05 pmol | 312 bp (b2 insert) 0.1 pmol | 2,628 bp (nt 3710–6337) 0.05 pmol | – |
| b3 | 210/291 (LSP) | nt 3709/3710 (HEL/POL junction) | pSM389‐392 | 0.04 pmol | 3,709 bp (nt 1–3709) 0.05 pmol | 291 bp (b2 insert) 0.1 pmol | 2,628 bp (nt 3710–6337) 0.05 pmol | – |
| c1 | 120/201 | nt 5494/5495 (POL/CP1 junction) | pSM153‐162 | 0.04 pmol | 5,494 bp (nt 1–5494) 0.05 pmol | 201 bp (c1 insert) 0.1 pmol | 843 bp (nt 5495–6337) 0.07 pmol | – |
| d1 | 120/120 | nt 6211/6212 (3’ UTR after CP2 stop codon) | pSM47‐55 | 0.1 pmol | 3,105 bp (nt 1–3105) 0.05 pmol | 3,106 bp (nt 3106–6211) 0.05 pmol | 120 bp (e1 insert) 0.2 pmol | 126 bp (nt 6212‐6337) 0.2 pmol |