A 1.3-kb filler DNA fragment flanked by two BspQ1 restriction enzyme sites was inserted between the 5′mir and the 3′mir sequences. Once digested with BspQ1, the double-digested vector then has two incompatible 5′ overhang ends and can be easily isolated from any single digested vector, thus eliminating self-ligation background in later procedures. The digested vector is ready for the insertion of the shRNA sequence, which is generated by annealing two single-stranded oligonucleotides to form a small double-stranded insert with proper overhangs as described in the paper.
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