The second step of Silent Site Insertion involves the actual selection of landmark restriction sites. Your protein sequence is displayed with an asterisk under every tenth amino acid. Anchored pull down menus contain all pf the possible silent, unique insertions.
The Silent Site Insertion Module - Landmark Selection
Easiest method: You can make the computer select sites for you with the "Pick Sites For Me" button. Enter an amino acid interval and GeneDesign will select sites on that interval using the criteria you defined in the last screen. The program's choices will be presented to you using the same screen and you will have a chance to edit them. You can change the amino acid interval and have the program reconsider as many times as you like.
Manual method: You can make your own landmark selections. Each pull down menu is anchored by a line and a dot between a pair of amino acids; these only appear where a silent mutation is possible. The pull down menus are populated with lists of enzymes that can be introduced at those positions. A black line indicates that none of the enzymes in the list are absent from the vector you selected (or present in the list of sites you created). A red line indicates that there are one or more enzymes in the list that are absent from the vector. Small warning: the program does not currently keep a list of your choices during manual selection, to help you keep track of which enzymes have been used already. We are working on this.
If you have the computer select the sites it will use blue lines to indicate its selections. If you like you may edit the selection of sites after the computer has made its selections, for example, if you really want a unique site at a specific position, or if you require a XhoI site somewhere in your gene. The next screen will warn you of any difficulties in landmark insertion.
Silent Site Insertion Module - Summary of Sites
Once you are satisfied with the sites that have been selected you can click the "Continue to Summary" button. The next screen will provide you with a detailed list of the sites chosen for silent insertion. The first column is the site name. The next column is the recognition sequence, followed by an asterisk if the enzyme exhibits star activity. The third column is the nucleotide position of the site. The fourth column defines the end type the enzyme will leave, 5', 3', or blunt. The fifth column is the incubation temperature of the enzyme. The sixth and seventh columns provide a general guideline to methylation sensitivity. The eighth column is vendor availability and the ninth column is the cost per unit in '04 dollars.
At the far left, there is a checkbox next to each enzyme name. If you used automatic site selection and are unhappy with one or more of the enzymes chosen, you can check any number of them and hit the "Reconsider" button. Automatic selection will be run again and the enzymes you select will be excluded from consideration.
Your new nucleotide sequence is at the top of this last screen. It would be wise to double check it
in your favorite sequence analysis program to be sure it produces
the correct amino acid sequence at this stage!
You can now take this sequence to another
module. To follow the "Design a Gene" path, click "Oligo
Design".