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. 2014 Oct 10;9(10):e110404. doi: 10.1371/journal.pone.0110404

Figure 1. SIM cassettes and GLVs.

Figure 1

(a) Schematic representation of SIM cassettes. SIM cassettes are classified into three groups based on the order of the use. Abbreviations and name of each cassette are shown. (b) A GLV was constructed by inserting a SIM cassette into a gene of interest (GOI)-encoding vector. For the sequential integration, first step is the introduction of a GLV having cassette 1 to the gene-loading site of the HAC using Cre recombinase. The cassette 1 contains Bxb1 attP sequence so that a GLV with cassette 2 can be integrated into the HAC as a second GLV by the use of Bxb1 integrase. φC31 attP sequence of the cassette 2 enables loading of the third GLV with cassette 3 using φC31 integrase. Afterward, alternative use of GVLs having cassette 2 or cassette 3 allows for sequential introduction of GLVs into the HAC. For the simultaneous integration, GLVs having cassette 1, 2 or 3 were loaded to the HAC simultaneously by using Cre, Bxb1 integrase, and φC31 integrase. For the combination of simultaneous and sequential integration of GLVs, cassette 1 containing FRT sequence was used. The FRT sequence makes it possible to integrate a fourth GLV having cassette 1 after the simultaneous integration of three GLVs. Fifth and more GLVs could be introduced in the same way based on the sequential loading procedure.