Plasmid pFUGW was constructed by inserting the following into the multicloning site of HR’CS-G: HIV-1 flap sequence PCR-amplified from the HIV NLA4.3 genome, the human polyubiquitin promoter-C (gift of L. Thiel, Amgen), the EGFP gene, and the WRE (woodchuck hepatitis virus posttranscriptional regulatory element) (gift of D. Trono, University of Geneva). Lentiviruses can be produced by cotransfecting the HIV-1 packaging vector Delta8.9 and the VSVG envelope glycoprotein into 293 fibroblasts.
Order of elements: CMV LTR PstI flap PacI Ubiquitin promoter SpeI HindIII PstI SalI XbaI BamHI SmaI KpnI GFP NotI EagI XbaI EcoRI EcoRV HindIII ClaI WRE ClaI SalI XhoI KpnI 3’LTR ApaI PmeI.
Please note that there are 2 gaps upstream of the EGFP between author’s provided sequence and Addgene’s quality control sequence. These gaps are in the non-coding vector region and should not affect the expression of EGFP.