Using Cloning->Create Fragment, I have constructed a fragment (from annotated part of sequence) with 5' and 3' additions ("overhangs") containing restriction endonuclease sites (plus terminal 4 nts for enzyme foothold), but do not get a digest report after selecting the created fragment and using popup "Analyze->Find restriction sites."

Is it about missing qualifiers in the fragment annotation? Does the fragment not meet a digestion criterion?  What am I doing wrong?

Okay, let me give you the real world problem so that you can see why just adding a single stranded DNA overhang to the termini won't work unless the annotated fragment double-stranded sequence itself is edited to include the extended site.

The user wants to create a fragment from the sequence coded by NCBI Nucleotide accession number CAA31046 (CD20).  The user wants to insert it into pUC19 (AccNum L09137) and decides to use the AvaI and SalI sites in the cloning multisite region of the vector, which means the user needs to put AvaI and SalI on the 5' and 3' ends of fragment CAA31046, which will be done in a PCR amplification.

The following sense (forward) and anti-sense (reverse) primer pairs are used to incorporate the sites:

sense:  5'-TGAATCCCGGG-[first 5' 15 or so nts of CAA31046]-3'
  where 5'-CCCGGG-3' is an AvaI site

anti-sense:  5'-AATAGTCGAC-[first 3' ?? nts of CAA31046]-3'
  which is  5'-GTCGACTATT-3' on the sense strand where 5'-GTCGAC-3' is a SalI site.

(The exact length of primers has been set so Tm is within 1-2 deg of each other and that 3' of primer is a C or G.)

So when the amplification is finished, the following amplicon is hopefully obtained:



and after the double digest the following is ready:



Since Create Fragment, then Digest won't work, the way I see it, the following must be done:

1.  The user must look at the double-stranded extensions to the annotated sequence from which a fragment must be created, perform the digest "on paper" and determine which of the extended parts remain double-stranded and which bases are single-stranded as a result of the digest (for enzyme not creating blunt ends).
2.  The double-stranded sequence to the fragment is then edited to include the double-stranded extensions.
3. Now Create Fragment can be used with the edited annotated sequence, and the overhangs representing the digest done.

My only question is if step 2 can be done (not tested by me yet).

If functionality can be created such that in the Create Fragment process, the user can specify 5' and 3' base pair (double-stranded) additions and then subsequently use the digestion function, that would seem useful.