PT2/C-FLuc, pT/CMV-SV40-LgT, pT/CAGGS-NRASV12 and PGK-SB13 were created as previously described(26 (link)). PT2/C-Luc//PGK-SB13 was created by excising the PGK-SB transposase expression cassette from pPGK-SB13 as a Xmn I/Pme I fragment and ligating into pT2/C-Luc as a Xmn I/Pme I fragment. PKT2/PGK-Bsd:GFP CLP-Luc was a kind gift from Andy Wilbur (University of Minnesota, Minneapolis, MN, USA). PLXIN-EGFRvIII containing the human EGFRvIII cDNA was a kind gift from Dr. Michael J. Ciesielski (Roswell Park Cancer Institute, Buffalo, NY, USA). PT3.5/CMV-EGFRvIII was created by subcloning EGFRvIII from pLXIN-EGFRvIII into litmus 29 (New England Biolabs) as a Spe I fragment, followed by ligation into pT3.5/CMV-GFP as a Xho I/Age I fragment. MSCV-LTRmiR30-SV40 (27 (link)) contained a microRNA short hairpin against Trp53 and a second expression cassette encoding GFP; it was a kind gift from Dr. Scott Lowe (Cold Spring Harbor, NY, USA). The shP53 and GFP expression cassette-containing fragment was released from MSCV-LTRmiR30-SV40 as a Pvu II fragment and ligated into PT2/HB (28 (link)) as an EcoR V fragment to generate pT2/shP53/GFP4. The MSCV-AKT vector was a kind gift from Dr. Scott Lowe. MSCV-AKT was cut with EcoR I/ Nco I to release the AKT cDNA and ligated into Litmus 29, followed by final ligation into pKT2/CLP as a Nco I/Bgl II to generate pKT2/CLP-AKT. Plasmids were purified using a maxiprep kit (Invitrogen) and stored in 0.1X TE buffer (pH 8.0, from a 1X stock solution comprised of 10 mM Tris-Cl and 1mM EDTA).