Part:BBa_K929103

B-cell transmembrane region with mCherry and TEV site, framed by LoxP

B-cell transmembrane region with mCherry and TEV site, framed by LoxP
BioBrick Nr.	BBa_929103
RFC standard	RFC 25
Requirement	pSB1C3
Source	Gene synthesis by GeneArt
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

This part represents the switchable membrane anchoring region of our antibody construct (BBa_K929107).

TEV recognition site:
The TEV protease cleavage site permits the shift from surface presentation of the nanobody-Fc fusion protein to a secretory 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.

LoxP site:
The membrane anchoring region is flanked by two LoxP sites introducing a non-reversible genetically switch to our system which allows the modification from surface presentation to secretion on the DNA level. Both LoxP sites are oriented in the same direction leading to the elimination of the enclosed region by cre recombinase activity.

Transmembrane domain of B-cell receptor with glycine-serine linker: modified BBa_K157010
Helical single-span transmembrane domain of the B-Cell-Receptor with a flexible 3 aa linker at the C-terminal end. 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.

mCherry:
Expression of the switchable membrane anchoring region and its cellular localization can be monitored by the mCherry signal with an excitation at 587 nm and an emission at 610 nm.

Characterization

For characterization and further applications see BBa_K929107.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]