OK this will be a little bit long-winded. Sorry. here goes
So first things first, you need to check which of the sites in the destination vector (and thus which sites you will adding to your primers) are NOT in the sequence itself. If they are then you will be cutting your gene of interest at the same time as you digest the sites in your primer ends (which is bad). In this case, you have the following enzymes present (see Fig. 1 graphicy thing): EagI, PstI, BamHI, ApaI, NdeI and ClaI. Meaning that you can only use the following: NheI, EcoRV, SalI, and MluI.
Next best step is to plan ahead. You’ll want to do a double digest (with the enzymes whose sites are put into your primers) so it helps if you see which of the available enzymes are compatible buffer-wise (NEB has an app that lets you find this or you can find it online. If you are using another vendor, this info should be available for their products as well). In your case, I took NheI (NEB# R3131S) and EcoRV (R3195S) because both come from NEB in the “High Fidelity” form that use the same buffer.
Now you’re going to make your primers. In this case the sequence is somewhat chosen for you since you need the whole thing. HOWEVER, your sequence that you gave starts at the ATG (start codon) of your GOI. This means that your PCR fragment will lack the Kozak sequence needed to start transcription. You want this section of your primers to be 20+bp (as Bob mentioned). I took the first 20 because the Tm (which will be used in your PCR) is already pretty high at 68C. Then you add a Kozak consensus sequence (italics) and the restriction site (underlined). EcoRV will go on the forward primer and NheI on the reverse so that when you ligate, your EGFR goes into the vector in the correct orientation. In this case, I will use the endogenous Kozac (lowercase, italics) as part of the primer. This will be your Fwd_Primer.
The reverse primer, I took ~30 to as to try to match the Tm a bit at 62C. The second has more As and Ts in that region, which is why the Tm is so low. Remember that this is going to be the reverse strand sequence. The gene-specific part is uppercased below. Then you have to add the site you want to use so the NheI is on the left (uppercase and underlined).
On both, add a few spacer bases so the enzymes can cut efficiently.
Fwd_Primer: gggggg GATATC cggggagcagcg ATGCGACCCTC Tm = 69C
Rev Primer: gggggg CATATG TCATGCTCCAATAAATTCACTGCTTTGTGG Tm = 62C
After you PCr and clone this, sequence, sequence, sequence! Your sequence has a LOT of repeat elements in it which may be incorrectly copied (see Fig. 2 graphicy thing), even by high fidelity polymerases so you will want do design sequencing primers within the EGFR so that you can sequence the whole thing!
Edited by Bio-Lad, 12 August 2014 - 10:13 AM.