A type-II homodimeric restriction endonuclease—popularly referred to as bagel two—which cuts DNA in a staggered manner wherever it finds the sequence AGATCT.
As done previously for VEEV [38], WEEV and EEEV DNA vaccine plasmids were then constructed by inserting the synthesized codon-optimized genes into the NotI and BglII restriction sites of the eukaryotic expression vector pWRG7077 (PowderJect, Madison, WI), which has been described previously [40].
A 1.7 kb fragment of mouse centromeric minor satellites was amplified from genomic mouse DNA with primers 5-AAA AAA AAG GAT CCA AAA TTT AGA AAT GTC CAC TG-3' and 5-AAA AAA AAA GCT TAA GAT CTC CAT ATT TCA CGT CC-3' and cloned into pBeloBAC 11 into BamH1 and BglII sites.
To generate luciferase reporter, the sequence of MMP-7 containing MluI and BglII restriction site was cloned into the upstream of firefly luciferase gene in PGL3 vector.
To facilitate cloning into the vector pCAMBIA1301, a restriction enzyme site of PstI was incorporated into a forward primer, whereas a BglII site was flanked with a reverse primer.
Twenty-one alleles of the IL6 promoter (690 bps, chr7:22732707-22733396) that contained 9 rs1800797 A alleles and 12 rs1800797 G alleles were successfully amplified using primers listed in Supplemental Material, Table S2, from 13 human BEC cultures that were heterozygous for rs1800797 and were directionally cloned into the pGL2-basic luciferase reporter vector (Promega) upstream of the luciferase coding sequence using MluI and BglII cloning sites according to the manufacturer's instructions.