|
1. Start GCK and open construct#2 from the tutorial files folder provided.
2. You will finish annotating this construct in this tutorial and learn how to define open reading frames (ORFs). First, choose Construct > Display > Show Construct Title which will place the title of the construct and the length of the construct into the center of the window.
|
|
Figure 11: Finding Open Reading Fames
|
3. Choose Construct > Features > Find Open Reading Frames... to bring up Figure 11. This dialog gives you complete information about how the program will find ORFs. If you are looking for ORFs in exons you might want to turn off the Start at 'ATG' check box - the program will then find any open reading frame (not just those starting with ATG). After you've adjusted the dialog to look like the one shown here, press the OK button to find the ORFs.
|
|
Figure 12: ORFs are Marked
|
4. After running the ORF search you will see two new objects in the construct window: arrows representing the two different ORFs found by this search, as shown in Figure 12. Both ORFs are highlighted. These ORFs represent regions of interest (regions). In addition to site markers and DNA segments, regions are a third kind of object that can appear in the construct window. Regions can be formatted using the Format > Regions > submenu.
5. Click somewhere in the window to deselect the regions just created. Click once on the region on the right side that overlaps the polycloning site. Choose Construct > Get Info... to bring up Figure 13. You can change the direction of the region by clicking the Top Strand or Bottom Strand radio buttons. The Protein Sequence check box determines if the region is to be translated or not when viewed as an actual sequence (see Tutorial 4: Viewing the Construct as a Sequence).
|
|
Figure 13: Region Get Info Dialog
|
6. This region is the beta-galactosidase region, so type beta-galactosidase into the Region Name text box. You can also enter comments into the Region Comments text box. Both the name and the comments can be searched by GCK later. In this case it might be reasonable to enter some comments about this vector and how the polycloning site disrupts the beta-gal coding region. Click OK to close this dialog box.
7. With the region still selected, choose Format > Regions > Show Region Names to display the region name in the construct window. You can select this name and alter the appearance of the text by using appropriate items under the Format menu. The region name text can be dragged around the construct window.
8. Now select the left region by clicking on it once with the mouse. Press command-I (Mac) or Ctrl-I (Windows) or choose Construct > Get Info.... Change the name of this region to 'ampicillin resistance' and put in comments indicating that the gene product is 'b-lactamase'. Click OK.
9. Note that when you have a region selected, you have access to many items in the Format menu. This will allow you to change the appearance of the arrow in the construct and even change directions by using the Format > Lines... menu. This would be equivalent to changing the protein direction in Figure 13.
|
