Plasmid construction

To construct pLV/AAVS1-donor-fluc-PGK and pLV/AAVS1-donor-egfp-PGK, inserts were amplified from pLV/fluc-PGK-PBT (Cai et al., 2014b) and pLV/egfp-PGK-PBT (cloned similarly as pLV/fluc-PGK-PBT), respectively, using primer sets YJ219F-YJ210R (primers are listed in Supplementary file 2A). The inserts were subsequently digested by BsiWI/SalI and cloned into pLV/AAVS1-donor-LS (Cai et al., 2014a) that was digested with the same pair of enzymes. pLV/AAVS1-donor-puro-PGK and pLV/CCR5-donor-puro-PGK were cloned similarly by amplifying inserts from pLV/puro-PGK-PBT using primer set YJ262F-YJ210R and ligated with pLV/AAVS1-donor-LS and pLV/CCR5-donor-LS (Cai et al., 2014b), respectively. Restriction enzymes used for inserts and vectors were BsrGI/SalI and BsiWI/SalI, respectively. All donor sequences are provided in Supplementary file 3. The packaging construct, pMDlg/pRRE-D64V-218, for production of IDLVs was constructed by introducing a D64V mutation into the integrase of pMDlg/pRRE by overlap PCR. For the first round PCR, primers AgeI-int(s)-int-D64V(as) and int-D64V(s)-AccIII-int(as) were used, respectively, with pMDlg/pRRE as the templates. For the second round PCR, primers AgeI-int(s) and AccIII-int(as) were used with the PCR product from the first round as template. The inserts and vector pMDlg/pRRE were then both digested by AgeI and AccIII before ligation.