Table 2.
Primers for the construction of CS-containing 7a, 8a and nsp-1 plasmids
Name of plasmid | Sequencea,b | Sense |
---|---|---|
pXJ-CS-7a | 5′-CGGGATCCTCTCTAAACGAACATGAAAATT-3′ | Forward |
pXJ-CS-7a | 5′-CCGCTCGAGTCATTCTGTCTT-3′ | Reverse |
pXJ-CS-8a-HA | 5′-CGGGATCCTCTCTAAACGAACATGAACCTTCTC-3′ | Forward |
pXJ-CS-8a-HA | 5′-CCGCTCGAGTTGTGTTGTACC-3′ | Reverse |
pXJ-CS-nsp1-HA | 5′-CGGGATCCTCTCTAAACGAACTTTAAACCATGGAGAG-3′ | Forward |
pXJ-CS-nsp1-HA | 5′-CCGCTCGAGTTACCTCCATTGAGCTC-3′ | Reverse |
These primers are used to construct insert than can mimic the CS-leader-containing full-length genomic mRNA (i.e. mRNA 1) and the subgenomic mRNA 7 and 8 found in SARS coronavirus infected cells (Yount et al., 2003). The highly conserved consensus sequences (CS), which are highlighted in bold, form the junction between the leader RNA sequences and the body sequences that encode the different open reading frames. The sequences that corresponded to the targeted sequence of siRNA 7 (TCTCTAAACGAACATGAAA) are underlined and the number of nt match by pXJ-CS-7a is 19 out of 19, by pXJ-CS-8a-HA is 18/19 and by pXJ-CS-nsp1-HA is 17/19, respectively.
The restriction sites used for cloning purposes are shown in italics.