2)Do (+ 98) degree 5 min with or without formamide prior loading of your plate to the instrument (sequencing).

can you cut it out of the original vector and re clone and sequence the new homozygous clones. Is it that you have 2 different clones in your dna preparation and sequencing other clones will give the answer. Can you post an original electropherogram so that we can see what the sequence looks like please?

Thank you for your suggestion.

DR. Paul Rutland, I cut a fragment and insert it another vector, but end of the fragment have a Tnos site and a reverse orientation Tnos also present one end of the vector. My DNA fragment is a PCR product, So for confirm I have to read this fragment.  

it sounds like your template is self annealing to form a double stranded region which is hard to sequence. If this is so try sequencing in the presence of 1M betaine just like doing PCR too help break the secondary structure and increase the extension and annealing temperature of your sequencing reaction by 2 .if your sequencing primers will still anneal at the higher temperature

Thank you Dr. Paul Rutland

if the sequencing still does not work can you design primers to sequence from the vector overlapping into your sequence but not including both nos sites then pcr and sequence each end of your sequence separately

Now I sequenced and read this segment by restriction digestion.  I found a restriction site between two Tnos.