Gene Construction Kit provides many different ways for you to annotate your construct’s appearance and mark important features.

1. Start GCK and open the file construct#4 that you saved in the previous tutorial. If you do not have this file, you can start with construct#4 from the tutorials folder we provided.
2. It is possible to modify the sequence appearance using many of the items in the Format menu. Select the AT -rich segment from nucleotides 10-33 by dragging with the mouse. Now Choose Format > Font > Times and notice that even though Times is not a monospaced font, GCK still spaces the characters so that each character occupies the same width along the line. Now choose Format > Style > Bold. The bold characters are wider than the non-bold characters so this part of the sequence becomes more “spread out”. You can adjust for that by choosing Format > Style > Condense, which condenses the spacing between characters. You can change colors, fonts, and styles for any segment of the DNA to help indicate certain features. Feel free to try some of these on this segment now.
3. Another method can be used to illustrate certain features. This is through the use of Frames. To see how frames work, select nucleotides 676-699 (they are in the polycloning region). Choose Construct > Features > Make Frame . This will create a frame around the segment of DNA you selected and will include the region (amino acid sequence) and site markers. It should look similar Figure 2.19. The frame is selected (highlighted) and encloses all information related to that particular segment of DNA.

   Figure 2.19: A Framed Sequence

4. The appearance of the frame can be altered by using items in the Format menu, as usual. Note that a frame must be selected (it must appear highlighted in the window) in order for items in the Format menu to have any effect on the frame itself. With the frame selected, choose Format > Lines > and choose the second line thickness in the menu (it is two pixels wide). Choose Format > Color > Yellow, and then choose a fill pattern using Format > Fill . You can try different patterns and colors to see how they look. (If you plan to print the sequence, remember to use a light color for the fill pattern so that it does not obscure the text.)
5. Note that the frame encompasses the site markers, the DNA, and the region. You can limit what is enclosed in the frame by choosing Construct > Features > Set Frame Contents… as shown in Figure 2.20. This dialog allows you to define which items will be enclosed in the frame. Set your dialog box to look like the one shown here and then press OK.

   Figure 2.20: Set Frame Contents

6. It is somewhat difficult to distinguish between the DNA sequence and the protein sequence, so let’s change the appearance of the protein sequence. Click once to select the protein sequence and then choose Format > Font > Times , then Format > Style > Italic . Your display should now resemble that shown in Figure 2.21. The ability to alter the display of the sequence in so many ways provides a great deal of flexibility in pointing out specific features of any sequence with which you are working.

   Figure 2.21: A Modified Framed Sequence

7. Choose Construct > Display > Display Graphics . Note that the new region you defined as the origin of replication also appears in the graphical display and is selected as it was in the sequence display. There is a direct correspondence between the two views of the construct. Changes you make in the graphical view appear in the sequence view and changes you make in the sequence view appear in the graphical view. The graphical view just represents an easier way to visualize your construct. For cloning fragments (Tutorial 6: Cloning a DNA Segment and Silent Mutations), this is essential to maintaining an easy to use interface.
8. Click on the BamHI site marker name and then select all site markers by pressing command-A (Mac) /ctrl-A (Windows) or by choosing Edit > Select All. We want to change the appearance of all the site markers except for the BamHI site so we need to deselect the BamHI site while leaving the others selected. This is done in the standard way of holding down the shift key and clicking on the BamHI site (this process is called shift-clicking). Make sure that you have all the sites selected with the exception of the BamHI site. Now choose Format > Site Markers > Show Site As Symbol. You have something resembling Figure 2.9C. Note that you might not have the same set of symbols as shown in this figure.
9. [NOTE – if working with the demo version of GCK, saving is disabled. If you plan on continuing on to the next Tutorial, you can leave the file you just created, construct#4, open. If not, close this file without saving the changes.]
10. If working with a ‘Trial Version’ or the ‘Full Version’ of the application, choose File > Save As… , name the file myconstruct#5, 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. That tutorial will explain how to use your new vector as the recipient of foreign DNA.

You can quit now, or continue on to Tutorial 6: Cloning a DNA Segment and Silent Mutations. If you plan on continuing, you can leave the file you just created, construct#5 , open. If not, close this file.