Coding

Part:BBa_K209402

Designed by: Katja Kolar   Group: iGEM09_UCSF   (2009-10-02)

AarI A-B part, signal sequence-FLAG-YFP [SSFYFP]

GPCR N terminal tags flanked by AarI AB sites.

Usage and Biology

Intended to be used as an N-terminal addition to G protein-coupled receptors. The signal sequence from prolactin is reported to increase surface expression of GPCRs and is cleaved off leaving the free FLAG epitope, which is recognized by the M1 antibody. The signal sequence is also necessary for the YFP (in this case Venus) to be translocated across the plasma membrane.

Documentation and characterization

We did not conduct a specific comparison of GPCR expression with and without the signal sequence. However, the signal sequence was used for a number of receptors we tested in our GPCR screen, described here: http://2009.igem.org/Team:UCSF/Navigation

Specifically, constructs containing N terminal YFP were functional, which may not be possible without the presence of a signal which allows the N terminus of the GPCR to be exposed.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 346
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 346
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 128
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 346
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 346
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 84


[edit]
Categories
Parameters
n/aAarI A-B part, signal sequence-FLAG-YFP [SSFYFP]