The second step on the Design a Gene path is the Silent Site Insertion module. This is where you will choose the vector you plan to use to carry your synthetic gene. Alternatively, you can define a list of restriction enzymes to be considered for insertion. Vector choice is an extremely important step in the process. The purpose is to instruct the program on what restriction sites you DO NOT WANT to use as special "landmark restriction sites" in your synthetic sequence, namely those already present in the vector. If you choose a very large vector with many sites in it, the program will not have any sites left to play with. Therefore, if you have a choice, use the smallest vector available to you. In practice pUC18/19 work well; derivatives lacking the multiple cloning site work even better.
Your input amino acid and first-pass (optimized) nucleotide sequences will appear at the top of the screen.
The Silent Site Insertion Module - Vector Selection
Next you must choose the sites to be inserted. If you specify a vector sequence, all sites in the vector are automatically removed from consideration as landmark restriction sites by the program. There are three ways to choose eligible sites. You may (A) simply select the sites from a list. Click on the site name in the left column and then the arrow button at the bottom of the column to move that site to the right hand column. Repeat the process on the right side to remove it. Only sites in the right column will be considered. Or you may (B) select the name of the vector you will be using from a pull-down menu. GeneDesign has the sequences of these vectors and will determine which enzymes are absent from this vector automatically for you. Lastly, you may (C) provide a vector sequence and a name for that vector. GeneDesign will parse the sequence and determine which enzymes are absent from your vector.
You may in addition specify a list of enzymes that must not appear in the
final sequence. (D) Click on the site name in the left column
and then the arrow button at the bottom of the column to move that site to
the right hand column.
Repeat the process on the right side to remove it. Only sites in the right
column will be processed. The sequence will be searched for these sites and
if they are found in your first-pass synthetic sequence, they will be removed. These
sites will not be considered for landmark restriction sites.
At (E) you can define the ranking criteria for sites to
be inserted. We have found the default criteria to be more than adequate for
most purposes. For more information about ranking enzymes, see Enzyme
Choosing.
Once
you have selected the vector and/or the sites you can click "Next
Step"
to move on to the next step in site
selection.