Translational_Unit

Part:BBa_K2090001:Design

Designed by: TP_CC-SanDiego 2016   Group: iGEM16_TP_CC_SanDiego   (2016-10-14)


phoA->LbCHI32->Flag tag


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 613


Design Notes

Signal sequence peptides allow for secretion of linked proteins located downstream of the signal sequence; as such, the phoA signal had to be designed upstream of the LbCHI32. The FLAG tag must flank the 3' end of the genetic sequence for proper location on the translated protein for immunoprecipitation. The stop codons on phoA and LbCHI32 were eliminated, and a stop codon was added downstream of the FLAG tag. Similarly, ATG methionine was eliminated from LbCHI32 and only retained in phoA to initiate translation at the phoA site.


Source

Alkaline phosphatase, or phoA, is a periplasmic protein in Escherichia coli that can be used by membrane vesicles in vitro for protein transportation.

LbCHI32 originates from the Limonium bicolor genome; the coding sequence was taken from GenBank: ACE79211.1.


References