OK. According to the sequence you pasted, your amplicon should be around 372 bp (124 aminoacids*3), so I´m confused about you saying you should expect a 600 bp amplicon?
Besides, now I see you are using degenerate primers (and since you lack nucleotide sequence info, these are REALLY degenerate). That could explain why you are getting an unexpected amplicon: it could be due to a simple mispriming.
Several strategies could help in minimizing off-target amplification. These are:
- "Hot-start" (keeping your tubes in ice, and placing them in the cycler block only after it has reached >80 degrees celsius).
- Raising your annealing temperature (Tann) an empirical number of degrees (2-5), or
- A "touch-down" protocol (starting with an annealing temperature which is ten degrees above your normal Tann, and lowering one degree per cycle through ten cycles (consult your cycler manual or brochure to do this) before letting the rest of the program cycle using the usual Tann.
Alternatively, you can redesign your primers.
The 3´end of your forward primer looks quite nice. A lysine in the 3´end is a good deal: only two possible codons coding for it (i.e. fewer degeneration). But I still would have continued to the tryptophan (only one possible codon) to enhance specificity. Thus, the sequence to add to the 3´end of your current forward primer would be: DSIUGG (Ser/Ala + Trp). I think this could enhance your primer specificity a lot.
With respect to the reverse primer, it looks like you´ve chosen many aminoacids with wide codon redundance. This greatly hampers your primer specificity.
I would use the following region to design a reverse primer:
gi 50593059 175 WSEDEL
Cdd:TIGR01323 172 MSEEQL
The REVERSE primer thus being:
I hope this helps at least a little.
Good luck with your experiments.