1. Start GCK and open the file construct#1 , which you created in the first tutorial. If you do not have this file, you can start with construct#1 from the tutorial files folder provided.
2. The next thing to do is to mark restriction enzyme sites that are located only in the polycloning region and nowhere else. To begin, double-click on the polycloning segment which has a blue lined pattern and is located at about 3 o’clock. You should see the segment become selected.
3. Choose Construct > Features > Mark Sites… . You will see Figure 2.8. This dialog is discussed more fully in “Marking Sites”. For now, make sure that the commercial (no isoschiz) list shows in the popup menu at the top of the dialog. A list of enzymes will appear in the left scrollable area. Press the Add All -> button to add all the enzymes in this list to the right side list of Enzymes to mark .

   Figure 2.8: Mark Restriction Sites

4. In the middle section on the left, set the numbers to match that in the figure so you will be specifying enzymes that cut more than 0 times but less than 2 times – i.e. unique cutters.
5. Finally, choose the middle radio button on the bottom left of the dialog box to specify that you want to see those enzymes which cut exclusively within the selected segment of DNA. Now press the OK button to mark the sites.
6. You will see the construct labeled as shown in Figure 2.9A. Note that all the sites are crowded together. While all the newly marked sites are still selected choose Format > Site Markers > Automatic Arrangement . This will rearrange the site markers so that they do not overlap resulting in something that looks like Figure 2.9B.

   Figure 2.9: Marked Sites

   A

   B

   C

7. As was the case for a selected segment of DNA, when a site marker is selected, you have access to items in the Format menu to change the display of the site marker. Now shift-click on the BamHI site to deselect it (all the other sites remain selected). Select the Format > Site Markers > Show Site As Symbol to change all of the sites to symbols except the BamHI site. Click on the BamHI marker and use the Format > Style menu to make the text bold; use the Format > Size menu to make the text 10 point. You should end up with something that looks like Figure 2.9C.
8. While the BamHI site marker is still selected, choose Construct > Get Info… or press command-I (Mac) or ctrl-I (Windows). This will provide you with the information shown in Figure 2.10. If you wanted to, you could actually change the name of the enzyme (not a good idea) or enter comments in the comments text box. The horizontally scrollable area in the lower part of the dialog box indicates the actual sequence at that location. The dotted red rectangle indicates the recognition sequence for the restriction enzyme and the two arrows indicate the actual cut sites for the enzyme. You can change the cut site by clicking on an arrow and dragging it. Press Cancel to close this dialog without saving any changes.

   Figure 2.10: Site Marker Get Info Dialog

9. Choose File > Save As… , name the file construct#2 , and save it in your own folder – please do not replace the tutorial files provided because others might want to use them later. You will need it again in the next tutorial.

You can quit now, or continue on to Tutorial 3: Marking Open Reading Frames. If you plan on continuing, you can leave the file you just created, construct#2 , open. If not, close this file.