The sequencing results showed that only the sequences on both sides of the target gene were present, and most of the sequences in the middle were missing. My multiple experimental results all point to the same missing fragment. Gel electrophoresis showed that the length of the target gene fragment was no problem. What factors may cause this problem? Could this missing sequence have been cleaved by DH5α competent cell?
What happens if you align your sequencing results to the original backbone plasmid sequence without your insert?
It may be that your template dna has 2 regions of homolory and forms a loop that the sequencing enzyme reads across so the target looks deleted. If your restriction digest of plasmid plus insert looks good then you could try linesarising your target with a restriction enzyme not in your insert and sequencing in presence of dmso up to 8% to minimise secondary structure issues. If you are using a commercial sequencing service then just linearise the vector with insert and request high GC sequencing. You can also sequence in presence of 1M betaine to minimise secondary structure but you will have to remove the salt very thoroughly as sequencers will load salt rather than dna if salt is available in the sequenced sample
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