I cloned my interested shRNA sequence in pLKO.1 neo - based lentiviral vector between Agel and ECOR1. After ligation when I did restriction digestion I got three bands with Nco1 and ECOR1 technically I should get two bands only. But, when I digested it with ECOR1 only I got the expected single band. AgeI was destroyed in all the clone.

I did sequencing with many samples almost every time I am getting first 4 base of my insert missing. In couples of times I got full insert but there was mismatch at the middle.

Could anyone suggest me is this problem in my vector or Oligos or  this problem may arise due to other things.

Regards,

Majid