Creating an expression construct

To generate a plasmid for expression of a recombinant protein can be challenging sometimes. In former times (prior to high fidelity polymerases) one had to find restriction enzymes that allow cloning of the coding sequence or its fragment into an expression vector. Expression levels were not always optimal due to the structure of the plasmid, not optimal Kozak sequence or some other reasons. Now generation of eukaryotic expression plasmids is really simple the strategy of this cloning can be divided into several steps.

1. Choose you favourite cloning vector. I usually use pcDNA3.1 it does relatively well in most of the cases.
2. Download the sequence of your gene from NCBI.
3. Locate coding sequence.
4. Identify restriction endonucleases that are present in pcDNA3.1 MCS and are absent from the coding sequence you want to clone.
5. Find start codon of your sequence (ATG). Check if there is a Kozak sequence around. In the best case it should look like accATGgnn, where ATG is your start codon.
6. If you have Kozak sequence, simply add a restriction site prior to ACC (for example GAATTCACCATGgnn for EcoRI) and order a primer that has 4 bases overhang, restriction site, gene sequence including Kozak, ATG and some coding sequence. The total length of 100% identical sequence should be at lease 15 at max 20.
7. If you do not have Kozak sequence, than simply add ACC in front of your ATG and GCG (Ala) immediately after the ATG. Then proceed as in the step 6 takin into account that your 100% identical part will start after GCG you added.

To be continued....