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One of the most useful features of GCK is called chronography. This feature allows you to keep track of construct history as well as to store different views of the same sequence in a single file. Chronography allows you the flexibility of displaying the same sequence in a number of ways to bring out key features.
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Figure 30: pBR322 as Graphic
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1. Start GCK and open the file called pBR322 in the tutorial files folder. You will see Figure 30. Note that the SalI site shows both the name and position for the marker. This can be set in the Format > Site Markers menu. The bottom right corner of the window shows a scale marker indicating the scale of the drawing. The scale marker can be shown or hidden by using the Construct > Display > Show/Hide Sites.
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Figure 31: pBR322 as Text
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2. Choose Construct > Display > Display Sequence to bring up Figure 31. This view of the sequence is a simple one, consisting just of the sequence grouped in tens and a few sites marked.
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Figure 32: pBR322 Generation#1
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3. Choose Construct > Display > Display Graphics to return to the graphical view. All the chronography features are accessible in the Format > Chronography submenu. Each different view of the construct is referred to as a different generation. Notice that at the bottom of the Chronography menu are three different named generations - restrictions sites, regions of interest, and DNA sources. These are the three different generations present in this construct. The current generation is checked. Choose Format > Chronography > Show Previous Generation. This will bring up Figure 32, which is one generation in the past and is referred to as generation#1. In this generation coding regions are shown along with an origin of replication.
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Figure 33: pBR322 Generation#1 Sequence
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4. Choose Construct > Display > Display Sequence to bring up Figure 33. Note that the sequence is now grouped in threes and the translation of the tetracycline resistance gene is shown. This is not a different sequence - it is the same sequence as before being displayed differently.
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Figure 34: pBR322 Generation#2
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5. Choose Construct > Display > Display Graphics and then choose Format > Chronography > Show Previous Generation again to bring up Figure 34. This generation shows the three different segments that were used to construct the original pBR322 plasmid.
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Figure 35: Segment Info
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6. Double-click on the blue segment and choose Construct > Get Info.... You will see Figure 35. Information can be stored about each DNA segment in the construct. By carefully documenting the sources of each segment, the entire history of a construct can be maintained. All the comments entered can also be searched using the File Searching (Tutorial 2) capabilities of GCK. Press the Cancel button to return to the graphical window.
7. Select the entire DNA of the construct by choosing Edit > Select All. Choose Format > Chronography > Restriction Sites. Remember, this is really just a different view of the same sequence you have been looking at throughout this tutorial.
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Figure 36: SalI Fragment of Actin DNA
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8. Choose File > Open and open the file called actin DNA, which is in the tutorial files folder. Double-click on the SalI fragment to select it (Figure 36), and then choose Edit > Copy. We will be cloning this fragment into pBR322.
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Figure 37: Actin Fragment into pBR322
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9. Put the insertion point at the SalI site on the pBR322 DNA. You can do this either by clicking in the DNA at the location of the SalI marker or by clicking on the SalI marker itself. Choose Edit > Paste to paste the actin DNA SalI fragment into pBR322 at the SalI site. You will get a warning that you are about to delete a region (the tetracycline region in generation#1 is there, even though you cannot see it - click OK) and then you will see Figure 37. Note that the positions in the SalI markers have been updated and the size of the construct has been updated in the center of the window.
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Figure 38: Actin Fragment into pBR322, Generation#1
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10. Choose Edit > Select All and then choose Format > Chronography > Show Previous Generation to bring up Figure 38. Note that the previous generation of the actin DNA was copied when the actin fragment was copied. All generations of a fragment of DNA are copied when that fragment is copied. This provides an easy way to keep track of where all the different pieces of DNA have come from when you assemble a construct of your own.
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Figure 39: Actin Fragment into pBR322, Generation#2
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11. Choose Format > Chronography > Show Previous Generation again to brings up Figure 39. Notice here that the actin fragment is just a plain gray line because there was no generation specified for the actin DNA that far into the past.
12. Choose Construct > Display > Display Sequence to view the construct as a sequence in generation#2. Notice how the gray from the actin fragment is also used to display the sequence itself.
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