ATGme

a simple DNA sequence optimization tool

Paste DNA sequence here:


Paste codon usage table here (available from http://www.kazusa.or.jp/codon/):


    
...

One-click optimize

Bulk optimize

Optimize by codon

Restriction sites

 

Check your optimized sequence for the presence of restriction sites.

Begin to type the name(s) of one or two restriction enzymes (for example, FatI or Cac8I). Matching enzymes will appear; click one to select it, or keep typing until only one matches. Then click "Check for restriction sites".

     
     

 
Alignment here

A few words before you start

Please note that this software is meant for a simple and fast way to identify rare codons and subsequently optimize your DNA sequence. The software does not address splicing motifs or ribosome binding sites which can be introduced in the process. Please consult other web-services for that.

Getting started

Input sequence

Paste the DNA sequence of the molecule/protein/ enzyme you want to optimize into this field in text or FASTA format. Numbers of any kind will be ignored.

Codon usage table

First, get the codon usage data from the Codon Usage Database by going to http://www.kazusa.or.jp/codon/. Type your organism into the search box and click submit to obtain the codon usage table of the organism of your choice. Please note that frequency values from CUD represent the mean values of the codon usage for all the genes in the Genbank files of a specific organism.

Copy the table (no headings, only table content) as it is (do not change the format!). For one codon this would look like this: “UUU 25.5( 217)”. You need to copy all 64.

Paste the table into the box under "Paste codon usage table here:"

Click the "Start processing" button to continue.

Once your sequence has been read and analyzed, you will see three new boxes at the top of the page:

Note that your sequence has not been optimized yet! You need to tell ATGme how you want to do that, using the three tabs under the "Optimized sequence" box.

Optimizing your sequence

One-click optimize

This is the most simplistic optimization possible: All codons in your original sequence are replaced with the corresponding redundant codon having the highest codon usage frequency in your chosen organism.

Just click the "Optimize" button. You will see the results in the "Optimized sequence" box above. The GC% and AT%, and the number of bases, for your optimized sequence are shown at top right.

Bulk optimize

This gives you a little more control than the one-click optimize, allowing you to exchange all instances of a certain codon for another codon.

The table lists all rare codons found, their usage per thousand and how often they appear in your sequence. Also here the color code (rare=orange, very rare=red) applies. Should you wish to see all codons, not just the rare ones, tick the "Show common codons" box above the table.

You can choose to exchange all instances of a certain codon with another codon simply by selecting it (see right column). The number next to the codon shows you how frequently it is used (in ‰). All codons of this kind will be exchanged with whichever codon you select (always encoding for the same amino acid).

As you work, you will see the results in the "Optimized sequence" box above. The GC% and AT%, and the number of bases, for your optimized sequence are shown at top right.

Please note that MUTATIONS are NOT possible.

Optimize by codon

Here you have full control, and can change each individual codon. Your sequence is shown in triplets; it is also translated and numbered. Just like the rest of ATGme, rare codons are highlighted in orange and very rare ones in red.

To optimize a codon, click on the drop-down list and select the desired redundant codon. All options are colour-coded according to their codon usage. Hold the mouse pointer over any of the options to see the usage in ‰.

As you work, you will see the results in the "Optimized sequence" box above.

Alignment

This box shows you an alignment of the protein sequences of your original and your optimized sequence. The DNA sequences were translated into the protein sequences according to the standard genetic code.

Identification of restriction sites

We recommend that you do this after you have completed your sequence optimization.

Open the tab called “Restriction sites”.

You can enter two restriction sites at a time. Begin to type the name(s) of one or two restriction enzymes (for example, FatI or Cac8I). Matching enzymes will appear; click one to select it, or keep typing until only one matches. Then click "Check for restriction sites" Write the name of the restriction enzyme (RE) as shown in the examples below so that the software will be able to recognize them correctly. If the enzyme does not appear it is currently not available for a search.

Examples:

The software can identify most of the commonly commercially available restriction enzymes. For an alphabetized list of enzymes check for example the website of NEB.

If the restriction site shows numbers in parentheses then these indicate the point of cleavage for non-palindromic enzymes.

For example, GGTCTC(1/5) indicates cleavage at:

If you found an unwanted restriction site, you can go back to the “Optimize by Codon” box and insert a silent mutation to change the restriction site into a sequence that cannot be recognized by your chosen RE. In the "Restriction sites" tab, if you hover your mouse cursor over the codon you want to change, you will see a tooltip with the codon number. Then you can find the codon more easily in the "Optimize by codon" tab.

Finishing up

Copy your optimized sequence into a document and save it. If you paste into Word, the rare/very rare colour coding in the Usage data, Original sequence and Optimized sequence boxes should be preserved.

To optimize another sequence, simply reload this page and start again.

Contact

Should you come across a problem, or if there is a feature you think we should definitely add since it would be invaluable for the scientific community, then drop us a line at: support@atgme.org

Please cite our paper!

©2015 Edward Daniel & Mirja Krause. All rights reserved.
This software is provided as-is, with no warranty whatsoever. You use it entirely at your own risk. We are not liable for any damages arising from your use of this software.