Part:BBa_K929101
Anti-EGFR scFv425 with TEV site, transmembrane region an eYFP
General information
| Anti-EGFR scFv425 with TEV site, transmembrane region an eYFP | |
|---|---|
| |
| BioBrick Nr. | BBa_929101 |
| RFC standard | RFC 25 |
| Requirement | pSB1C3 |
| Source | BBa_K157001, BBa_K929100, BBa_K157010, BBa_E0030 |
| Submitted by | [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] |
The Biobrick BBa_K929101 is an extended version of the Biobrick BBa_K929100 and is composed of the already existing human EGFR (ErbB-1) signal peptide (BBa_K157001), the scFv anti-EGFR-domain-3, a TEV recognition site (according to life technologies AcTEV TM Protease manual), the modified transmembrane domain: transmembrane domain of B-cell receptor with glycine-serine linker (BBa_K157010) and the enhanced YFP (BBa_E0030) reporter. The composite part was derived by PCR assembly.
Signal peptide:
The human EGFR (ErbB-1) signal sequence fused to the N-terminus of a fusion protein with a transmembrane domain mediates the transport to the membrane (BBa_K157001).
Anti-EGFR scFv425:
The single chain fragment (scFV425) specifically binds to the epidermal growth factor receptor (EGFR) domain 3. It has a shortened N-terminal FLAG-tag sequence of five amino acids (DYKDE) for purification and detection.
TEV recognition site:
The TEV protease cleavage site permits the shift from surface presentation of the scFv antibody to a secretory scFv production on protein level. The TEV protease recognition site ENLYFQG is the most commonly used aa sequence for recognition by the 27kDA catalytic domain of Nuclear Inclusion a (NIa) protein encoded by the tobacco etch virus (TEV). This sequence is extended by a 3 aa linker at the N-terminal and a 2 aa linker at the C-terminal end.
Transmembrane domain of B-cell receptor with glycine-serine linker:
Helical single-span transmembrane domain of the B-Cell-Receptor with a flexible 3 aa linker at the C-terminal end (modified BBa_K157010). This part was designed for fusion to proteins or peptides that will be presented on the cells surface. A signal peptide at the N-terminal end of the fusion protein for membrane integration (e.g. part BBa_K157001) is highly required.
Reportergene: Enhanced YFP ( BBa_E0030)
Expression of the scFv fusion protein and its cellular localization can be monitored by the enhanced YFP signal with an excitation at 514 nm and an emission at 527 nm.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 610
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3
| None |