Hi!

Making changes aruond RBS should be made with great caution.

What will affect:

1. SD-antiSD complementarity

2. SD-ATG distance (typically 5-7nt)

3. AT-rich site slightly upstream of RBS.

4. Sequence of spacer between RBS and ATG.

I would rather suggest to have optimal distance between RBS and ATG set at 8 nucleotides counting it as follows: GAAGGAGnnnnnnnnATG. When you shorten it to only five, it would certainly lower the efficiency of protein expression. Sequence of the spacer also matters, it should not make any funny secondary structures like hairpins etc. You may try to check the sequence of the RBS-ATG and nearby with mfold or similar RNA folding software for potential secondary structures there.

Dear's Victor G Stepanov and Anatoliy Zubritskiy

Thank you so much for your comment.

These are so useful for me.

You can also use the RBS Calculator model to predict how a sequence mutation will affect the mRNA's translation initiation rate. Go to https://salislab.net/software/predict_rbs_calculator. Copy/paste your mRNA sequence. Get a prediction for every start codon. For each prediction, it will calculate & show you the key interactions between the ribosome and mRNA.

You can also use the RBS Calculator to design a synthetic RBS to rationally increase or decrease the translation initiation rate.

Hello,

I suggest the sequence called the T7 phage g-10 leader sequence for high expression of your protein. However, right before the start codon, you can change the sequence to CAT. This will be a NdeI cut site. This change still shows high protein expression and is found in the pJL1 plasmid backbone.

After your promoter, the sequence will look like this:

gagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatatacatATG

https://www.addgene.org/102634/