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(1) Output file name

CSO creates an output FASTA file with your optimized DNA sequence, together with some other information about the optimization. In this field you can enter the desired name of this file (e.g. My optimized gene). There is no need to write the file extension.

*This file will be created by default in the directory where the app is running. If you wish to change this, you can give your file a name specifying the full path to where you want it to be saved (e.g. C:\Users\user\Desktop\My optimized gene).

• If a file with the name you specified already exists, the new data will be appended to it.

• If you leave this field empty, the default name of the output file will be ‘optimization_output’.

 

(2) Target amino acid sequence – obligatory field in all cases

This is your gene of interest, for which the sequence optimization will be carried out.

• Use 1-letter amino acid symbols (http://www.fao.org/docrep/004/Y2775E/y2775e0e.htm).

• Include the START (typically ‘M’) and STOP (‘*’) codons.

• You can either upload a FASTA file containing your sequence, or type / paste it in the field.

• Both lower and upper case letters are acceptable.

• The optimization will not begin if this field contains invalid AA characters, or if it is left empty.

 

(3) Organism – the default selection is C. reinhardtii nucleus

Select your required host. The two basic options are either C. reinhardtii nucleus (def) or C. reinhardtii chloroplast. Selecting between these two options will let CSO know which set of reference sequences to load and use. The third option is custom; selecting this option means that you want to use the CSO algorithms to design a synthetic gene for expression in a different host (e.g. a different alga/plant, bacteria, mammalian cells, etc.). If you select this option, input field (6) will appear and you will be required to provide a set of coding sequences from your host of choice that will be used as the algorithms’ reference sequences. We would like to note that this use-case will give you the general versions of the algorithms, without the C. reinhardtii specific optimization embedded into the two other use-cases. Still, performing sequence optimization in this manner is expected to yield decent results on average.

 

(4) Codon selection method – the default selection is most frequent codons

Select your algorithm of choice. The default option (most frequent codons) will simply give you the same codon (the “best”, or most frequent one) per each amino acid. This will result in a monotone (or “boring”) sequence with a codon adaptation index value of exactly 1, given the CSO reference set. The other options are:

A. Optimal codons and folding

B. ChimerMap

C. Mimic original host

For algorithm descriptions see the papers referenced from the buttons on the bottom left corner (relevant literature).

 

(5) 40 bases upstream from target gene – obligatory field only if it appears, otherwise it is hidden

This can be considered as the 5’ UTR region of your target gene. The sequence in this region is taken into consideration while selecting codons for your target gene.

• You can either upload a FASTA file containing your sequence or type / paste it in the field.

• Both lower and upper case letters are acceptable.

• If you provide more than 40 nucleotides, only the last 40 will be considered.

• The optimization will not begin if this field contains invalid DNA characters, or if the sequence provided is shorter than 40.

 

(6) Custom reference coding sequences – obligatory if ‘custom’ organism was chosen

Upload a FASTA file of coding sequences taken from the organism in which the user wishes to express his gene.

 

(7) Reference coding sequences from original host – obligatory if ‘mimic’ algorithm was chosen

Upload a FASTA file of coding sequences taken from the original organism that your gene of interest comes from.

NOTE – in order for the Mimic original host algorithm to work, one of these CDSs must code for the AA sequence you have specified in field #2.