During the use biobrick parts or biological devices (like a promoter), it is very limited to use the EcoR1, SpeI, XbaI, PstI enzyme sites for the insert gene. To compensate for this restriction, we’ve decided to use the primer design used in infusion cloning.

1. It is difficult to identify whether the Anderson promoter is ligated to the vector since the Anderson promoter is only 33bp. Thus, according to the other teams as well, the Anderson promoter wasn’t ligated into the 3a assembly and amplified. Instead, they’ve redesigned the oligo of the Anderson promoter(5' EcoR1 overhang---- 3' spe1 3'over hang) and used the annealing method. However, TNCR Korea used a different method in hopes of using the anderson promoters that enable gene expression control. If we use the infusion primer design and attach the Anderson promoter at the primer and go through PCR the Anderson promoter will naturally amplify. We can identify this with the PCR product bend test. This is because if the primer doesn’t work, we won’t be able to identify the bend.

2. Like DPP4, if there are EcoR1, Spe1, Xba1, Pst1 site which are used in 3A assembly, it is read as illegal insert gene which is unable to use. Thus, a step that changes a sequence like point mutation is required which is infusion cloning. Ligation utilized in infusion cloning only requires cutting a vector, not enzyme cut of the insert gene. This is because, insert gene naturally inserts itself into vector while it is amplified by PCR with primer sequence.