Generation of Pkt2-Luc-T2a-HPV18E7E6(del D70)

The generation of pKT2/CLP-AKT plasmid [32 (link)] and pCMV(CAT)T7-SB100 plasmid [33 (link)] has been described previously. These plasmids were purchased from Addgene. The generation of Pkt2-cMyc has been described previously [31 ]. To generate Pkt2-Luc-T2a-HPV18E7E6(del D70), 18E6 (delD70) was first amplified via PCR using the Pkt2-LucHPV18E7E6 [34 (link)] template and the following set of primers: 5′-CTGGCTCGAGGAGGGAAGGGGAAGCCTGCT-3′, 5′-GCTCCCGGATTCTGCTGTAGAAGATACACTTGTGGCAAGCGGCG-3′, 5′-CGCCGCTTGCCACAAGTGTATCTTCTACAGCAGAATCCGGGAGC-3′, AND 5′-AAACCAGCTAGCTGGTTATTACACCTGGGTCTC-3′. The amplified PCR product was then cloned into the Xho/bstX1 sites of a Pkt2-LucHPV18E7E6. Plasmid construct was confirmed using DNA sequencing and the DNA was prepared using an endotoxin-free kit (QIAGEN, Valencia, CA, USA).