My experiment requires using blasticidin selection marker in lentivirus transduction and I did cut the sequence of interest with restriction enzyme and insert it into the backbone carrying BSR (blasticidin resistant gene). The backbone previously worked very well, and with a concentration of 10ug/mL of blasticidin, I got my stable cell line. However, this time, the transduction seemed not to work and all the cells in titration were killed by blasticidin. I first thought the concentration too high for selection. Though I switched concentration of blasticidin to 5ug/mL, no cell in transduction group could survive, which suggested something more crucial, like plasmid design, might go wrong. Here is a short sequence showing the cut sites of both plasmids for getting the insertion part and BSR backbone. Both plasmids worked very well previously.

The yellow marked region is same in both donor plasmid and BSR backbone. (including Hind III site)

The insertion part has a longer gap between its ORF and IRES. The shorter gap shows region of BSR-carrying backbone. (including Not I site)

I cut them by Hind III and Not I, and after midi prep I verified the BSR marker is inside the ligated product.

The packaging region for lentivirus is within the length limit.

Protocol for producing lentivirus was same with my previous experiment wherein same backbone was used.

Does the gap length between 1st ORF and IRES influence the expression of 2nd ORF? Though the donor plasmid can well express its selection marker?