Part:BBa_K2170000
Biotinylated receptor with biotin acceptor peptide combined with a secretory BirA
Fusion protein with a Biotin acceptor peptide (BAP) for intracellular biotinylation by biotin ligase BirA. It's expression is regulated by a CMV promotor and its secretion guided by a BM40 signal peptide. For better characterization a mRuby3 was added to the intracellular part of the fusion protein. Also an A3C5-Tag (intracellular), an antibody binding site, as well as a Strep TagII, a binding sequence for streptavidin, was added to have different possibilities for later characterization of the transfected construct. The intr- and extracellular parts are connected by an EGFR TMD with stop transfer signal, which makes sure that the TMD stays inside the membrane. The Biobrick contains an internal ribosomal binding site in front of the BirA so that it, although it is part of the same mRNA, is translated seperately from the rest of the transcript. The fusion protein is directed to the cellmembrane where it presents the biotinylated BAP on the surface where it can be bound by free streptavidin to impart a polymerization reaction between the cells which makes the scaffold free printing even possible.
CMV_BM40_BaP_NanoLuc_A3C5_EGFR TMD_mRuby_Strep_polyA_IRES_Kozak_IgKappa_BirA_StrepDEL_polyA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 576
Illegal BglII site found at 3529
Illegal BamHI site found at 2597
Illegal XhoI site found at 761 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2372
Illegal BsaI site found at 4077
Illegal BsaI.rc site found at 743
Illegal SapI site found at 2529
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