Part:BBa_K3756010
CKLF1C19-Lamp2b-GATA3-siRNA-2
This DNA circuit is designed to express specific proteins and siRNA that specifically target TH2 cells and down-regulate the expression level of relevant target mRNA. It can express the fusion protein CKLF1C19-Lamp2b, where CKLF1C19 is a CCR3-targeting peptide, so it can specifically target to the surface of TH2 cells. Lamp2b is an exosomal membrane protein. Thus, fusion protein CKLF1C19-Lamp2b was able to target TH2 cells by exosomes secreted by cells expressing this protein. At the same time, the DNA circuit also expresses siRNA of GATA3, which can specifically degrade GATA3, an important transcription factor associated with type 2 inflammatory response in TH2 cells. Thus, cells expressing this DNA circuit could express siRNA of GATA3 and specifically target GATA3 in TH2 cells with the help of CKLF1C19-Lamp2b, thereby inhibiting its expression. Most importantly, the DNA circuit has attP and attB sites on both sides of the expression frame. In E.coli ZYCY10P3S2T expression system, Intergrase φ31 can loop out the sequence between attP and attB to form minicircle DNA. The plasmid skeleton will be cleaved by I-SCEL enzyme. (Kay MA, et al. A Robust System for Production of Minicircle DNA Vectors. 2010) In our project, the DNA circuit will be prepared in E.coli ZYCY10P3S2T expression system to form Minicircle DNA, and then the latter will be used to verify its therapeutic effect on asthma mice by transfection of mouse lymphoma cell line EL-4 and aerosol administration into mice. Details can be found in the project wiki.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1111
Illegal SpeI site found at 424
Illegal PstI site found at 808 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 424
Illegal PstI site found at 808 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1111
Illegal SpeI site found at 424
Illegal PstI site found at 808 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1111
Illegal SpeI site found at 424
Illegal PstI site found at 808 - 1000COMPATIBLE WITH RFC[1000]
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