I recently ordered a cloning product from Thermo Fischer, and the instructions for primer design require that the forward primer have the Kozak sequence ([G/A]NNATGG) on the 5' end. However, I have never designed primers requiring this sequence. How do I design the primer? I made two examples below, but I am not sure which one is correct:
1. GagatctgtcaagagaatccATGG
2. AAATGagatctgtcaagagaatcc
Hi Kristen Navarro , if the instruction os specifically to have Kozak at the 5' end I think they are both ot correct. [G/A]NNATGG) means:
position 1 is a purine, either a G or an A; position 2 and 3 can be and nucleotide; and positions 4-7 are ATGG (where ATG is the start of the protein). The sequence makes translation more efficient in some context, so
5'-GAAATGGNNNNNNNNNNNNN-3'
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