Design of expression plasmids using tools from custom vector suppliers

🧬Do you need an expression plasmid that is not commercially available (e.g. a mutated variant)?😏

Whether you want the plasmid to contain a mutated gene or a wild-type coding sequence, here is a quick step-by-step guide.📋

The first thing is to find the coding sequence of the wild gene that expresses the protein you want. To do this, go to https://www.ncbi.nlm.nih.gov/ and search the name of your interest gene using the Nucleotide search.

Then click on the desired organism and select mRNA. Choose the best entry for your query according to your requirement, and select the CDS (and the protein sequence will appear on the right, a.k.a., translation). 

Next, copy-paste both FASTA text codes into a Word file. We will need them later.

👉If you only want to express the wild-type protein...

Paste the CDS directly into the corresponding ORF item from the supplier (e.g. VectorBuilder). 

https://en.vectorbuilder.com/design/pRP_Exp.html

You will then only need to choose the promoter (usually a CMV) and a tag (fluorescent or not) that can be detected indirectly.

👉Do you want to express a mutant or synthetic protein? 

Click on your "/db_xref="CCDS:" to know the corresponding sequence(s) of the CDS to the amino acid(s) you want to change/remove (another web page will be opened, there, when you click on an amino acid, the corresponding genetic codon is marked in the nucleotide sequence).

Edit the sequence(s) in the Word file.

Use the genetic code to choose the codon whose nucleic bases translate your new amino acid(s).

https://www.genome.gov/genetics-glossary/Genetic-Code#:~:text=%E2%80%8BGenetic%20Code&text=Each%20gene's%20code%20uses%20the,each%20position%20within%20a%20protein.

Then, paste its CDS directly into the corresponding ORF gap of the website from the supplier.

J.