specific genes (cds or mRNA) sequences for a particular protien /enzyme/amino acid sequnce available in NCBI and other data bases. if you want work with aa sequences you have change it in cDNA sequence with help of bio informatics tools or have to go for the BLAST N to find out matching CDs or mRNA sequences in order to desigine primers.

Yes

Yes, it would be advisable to check NCBI or other data bases to see if the DNA sequences are available. If not, you will need to use sequence tools to design degenerated primers based on the amino acid sequence, taking into consideration of codon usage in a particular organism.

Yes, I have tried degenerate primers and meanwhile trying for the alternate. It is the codon usage cells that I have been using for my other studies and will be considered for this one too. Thank you very much for taking your time for this.

It is always highly reliable to track through amino acid sequences obtained on N-terminal sequencing and internal peptide sequence maps of mass spectrophotometry studies using purified protein or its sub-unit fractions. These amino acid sequences are normally aligned with sequences obtained from databases on a particular model or species or up to an order/family or group of similar such protein sequences of any source (even a mammalian model). If our peptide sequences are forunately aligned with both the ends of a known retrieved sequence, this would form a specific set of primers. Otherwise, it is only a degenerate set of primers. Generally, complete alignment in such situations is rarely possible. It may lead to designing of primers for RACE-PCR for fishing out the specific gene.

Yes Sir. Thank you very much. It is how I'm doing it now as per this and your previous suggestions.