TABLE 2.
Construction of plasmids to express μNS truncations
| Constructa | Enzymesb | Size (kDa)c |
|---|---|---|
| pCI-M3(1-713) | XhoI, NheI | 79.3 |
| pCI-M3(1-700) | XhoI, NheI | 78.0 |
| pCI-M3(1-683) | SalId, NheI | 76.1 |
| pCI-M3(1-470) | KpnI (blunt), SalI | 52.0 |
| pCI-M3(1-362) | KpnI (blunt), SalI | 39.9 |
| pCI-M3(1-221) | KpnI (blunt), SalI | 23.9 |
| pCI-M3(1-173) | SacI (blunt), SalI | 18.7 |
| pCI-M3(173-721) | EcoRI, SalI | 61.6 |
| pCI-M3(221-721) | EcoRI, SalI | 56.4 |
| pCI-M3(363-721) | EcoRI, SalI | 40.3 |
| pCI-M3(471-721) | EcoRI, SalI | 28.2 |
a
Each construct was designed to express a truncated μNS protein comprising the indicated amino acid residues.
b
Unless otherwise noted, pGEM-4Z plasmids from Table 1 were digested with the indicated enzymes to remove the truncated M3 genes. In some cases, one of the resulting fragment termini was converted to a blunt end (blunt) with T4 DNA polymerase before ligation to pCI-neo.
c
Expected size of the expressed μNS truncation.
d
pCI-M3(T3D)(1-683) was subcloned from pFastBac-M3(T1L)(1-683) as described in Materials and Methods.