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. 2016 Sep 20;44(21):10367–10376. doi: 10.1093/nar/gkw821

Figure 4.

Figure 4.

Functional mapping of the region required for tandem integration of the LE26 duplex by His-taged Cas1. (A and B) Integration of the 6-FAM-labeled LE26 duplex (200 nM) was monitored as described in ‘Materials and Methods’ section with either 35 or 70 nM His-tagged casposase and 1.5 μg/ml of plasmids harboring various deletions of the target segment borne by pMA-Target. The concentration of the casposase and the borders of each target site are indicated above each lane.A: gel scanned for 6-FAM fluorescence; B, same as A, after ethidium bromide staining. (C) Diagram summarizing the results obtained for the tandem integration of the 6-FAM-labeled LE26 duplex using the plasmids mentioned in Table 2. The TSD segment is shown in green. (D) Structure of the tRNA encoded by the region upstream of the Aciduliprofundum boonei TSD segment. Arrows indicate the position of the tRNA gene and its intron relative to the insert of pMA-Target and of features such as the beginning of the TSD or the beginning of the insert borne by the pMA-T103-140 plasmid. Numbering starts at the beginning of the insert borne by pMA-Target. The Figure was drawn using the software available online and described in ref. (45).