Part:BBa_K4173046
pcDNA3.1-CD63-L7Ae-mCherry
This DNA circuit is the most important plasmid which was desinged for the expression of fusion proteins CD63 and L7Ae, with (Gly)8 in between as a linker. CD63-L7Ae is a structure that promote the package of mRNAs into exosomes because of its location on the membrane and its interaction with RNA.CD63 is a member of the tetraspanin superfamily of activation-linked cell surface antigens with N-terminal and C-terminal intracellular domains, two link domains and two annular extracellular domains, and is one of the most widely used exosome markers(CD63, CD9, CD81). More recently, however, their presence in other EVs has been observed. According to the latest study, CD63 was proved to be the specific marker for exosome, while EVs bearing only CD9 or CD81 but not CD63 probably did not form in endosomes. CD63 is known for its abnormally high levels on the surface of activated basophils, on proliferating mast cells, and on the surface of endothelial cells in inflamed tissue. CD63 plays an important role in the regulation of cargo sorting and vesicle formation. As a transmembrane protein located specifically on exosomes, it can be used as a base protein for fusing other proteins on which help the target protein locate on the membrane and be packaged into exosomes. L7Ae is an archaeal ribosomal protein 7Ae 60S large ribosomal subunit, a subunit of archaeal RNase P, plays multiple roles in archaea. It is part of the H/ACA and the C/D box snoRNPs, which catalyze rRNA pseudouridylation and 2’-O-methylation respectively. Thus, L7Ae is one of the most commonly used riboswitches which RNA-only delivery gene circuits highly rely on except RNA binding proteins (RBP). L7Ae, a K-turn and K-loop binding protein, is a component of box C/D RNPs and interacts with the K-turn motifs of archaeal box C/D sRNAs3. Through the interaction between L7Ae and C/D box RNA structure which is added at UTR of reporter gene, the target protein can be grasped and stay close to L7Ae. Then target protein can be held on the membrane of exosomes when L7Ae is conjugated to the C-terminus of CD63. Researchers have designed many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins, and (Gly)8 designed by Sabourin et al. is one of flexible linkers which are usually applied when the joined domains require a certain degree of movement or interaction. Consisting of purely glycine, the small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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