Figure 1. The structure of Enzyme USB(AarI)
In order to easily and quickly insert the target function enzyme into our system, we design two enzyme-USBs. The enzyme USB have three fundamental components, flexible linker- enzyme adaptor-flexible linker. The first flexible linker has deleted the PstI recognition site. And at the beginning of the sequence there is a Bsu36I recognition site. The second flexible linker we replace the original PstI site with a isocaudamer SduI, since our part can not have a PstI recognition site. On the other hand, the enzyme adaptor has two same restriction enzyme recognition sites. In one of our enzyme-USB, it is the AarI recognition site; The other enzyme-USB is the BsmAI recognition site. The AarI and BsmAI are similar to BsmBI which all can make a 4bp sticky end designed by ourselves.
Usage and Biology
When we want to insert a functional enzyme into our fusion protein, first we need to have a PCR experiment to add a head and a tail around our enzyme. After that, the enzyme product also has the restriction enzyme recognition site. When digested by the specific restriction enzyme, it can generate the same sticky ends, so our enzyme can be inserted into our part.
The primer sequence:
primer1：5' GGAATTCCACCTGCAATTTGCT(E*20) 3'
primer2：5' GGAATTCCACCTGCAATTCTAA(E*20) 3'
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC