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1. Up to now, you have been working with a graphical view of the construct, but underneath all of this is a real DNA sequence. Start GCK and open the file construct#3 from the tutorials folder.
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Figure 14: General Info Dialog
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2. Choose Construct > General Info... to bring up Figure 14. The General Info is associated with the construct as a whole and is not attached to any specific part of the construct. It is a useful place to store information such as storage location for the DNA and other information as shown in construct#3. In combination with the File Searching (Tutorial 8) ability, the comments can be used to create a database of information on all your constructs.
3. Scroll until you see the information stating that the f1 DNA is in the segment from 3-458. What we would like to do now is indicate the location of this particular segment of DNA, but how do we define the locations in our graphical construct which does not have any markers at those locations? The answer is to view the construct as a sequence rather than graphically. Close the General Info window and then choose Construct > Display > Display Sequence. This will bring up Figure 15. If your display does not look like this figure, then scroll the sequence all the way to the top of the window. Notice the correspondence between the graphical view and the sequence view. Colors are maintained, sites are marked, and regions are indicated. However, since we specified that the regions we marked are protein regions (Figure 13), the translated peptide is displayed below the actual DNA sequence. The small triangles adjacent to the amino acid line numbers indicate the direction of translation.
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Figure 15: Viewing the Construct as Sequence
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4. Before we can mark the f1 origin of replication, we need to be able to identify the nucleotides to be marked. Add position indicators by choosing Construct > Display > Show Positions. Double-clicking to select a segment works the same way in the sequence view. Try this by double-clicking anywhere in the sequence to get a feel for how segments are defined.
5. With the insertion point anywhere in the DNA sequence, choose Edit > Select All (command-A on Mac, Ctrl-A on Windows). Now choose Format > Grouping > Group by Tens. Note that there is an option in the Format > Grouping menu to define your own group size.
6. Grouping by ten does not look right for viewing those parts of the sequence that correspond to translated regions. Double-click on the amino acid sequence corresponding to the b-galactosidase gene (it ranges from 244-846) to select the corresponding DNA and choose Format > Grouping > Group by Threes. This is a more appropriate grouping for coding DNA.
7. We now need to indicate our origin of replication. Identifying origins of replication is something that you might like to do in all of your constructs and you would like an easy visual method of identifying those regions from amongst the many that you might end up marking. You can do this by defining a specific color to represent an origin of replication. Choose Format > Color > Add a Color.... You will see Figure 16. Type in 'origin of replication' for the name of the color and then press OK. Choose a color from Apple's standard Color Picker to define a new color called origin of replication. This color will appear in the Format > Color menu. The ability to define your own color and to name it provides an easy way to indicate features of interest such as promoters, enhancers, and other sites on DNA.
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Figure 16: Add a Color Dialog
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8. Use the mouse to select nucleotides 3 through 458. You can confirm that you have the correct nucleotides selected by looking in the lower left corner of the window. Choose Construct > Features > Make Region.... You will see the dialog shown in Figure 17. Make sure you type in the correct name and put any comments you have into the comments area. This is NOT a protein sequence so do not check the Protein Sequence checkbox. Confirm that the first and last nucleotides are 3 and 458, respectively. Press OK.
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Figure 17: Creating a New Region
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9. The newly created region will be selected (if it is not selected, then just click on the region once to select it) so you can now alter its appearance. Choose Format > Lines and select the line having an arrowhead at each end. This will change the region to have two arrowheads.
10. With the region still selected, choose Format > Color > origin of replication to change the color of the region to indicate its function.
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Figure 18: Origin of Replication Indicated
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11. You should see something like Figure 18. You can now easily recognize the origin of replication by color and can use that region to select the corresponding DNA by double-clicking on the region.
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