Part:BBa_K3505023

FFAR2:V2tail:TCS GB compatible with B2-B3

Usage and Biology

FFAR2 is a naturally found eukaryotic GPCR protein that exhibits high affinity for SCFAs (acetic, propionic and butyric acid)(Kaemmerer, 2010) . After background searching through the NCBI database, we have identified the gene coding sequences needed for the designing of this gene construct. More specifically, a vasopressin receptor 2 segment has been attached to the C-terminal of the receptor, as it mediates recruitment of a TEV tagged b-arrestin-2, along with a TEV cleavage site(TCS).

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is presents in pUPD2 and has overhangs compatible for Golden Braid cloning. The CDS has position B2-B5.

Figure 1.The overhangs of this part in the Golden Braid Grammar.

Figure 2. The level 0 module : pupd2-FFAR2:V2tail:TCS (illustration from SnapGene)

Verification of Cloning

Fig.1:(U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204 bp, Positive Result:C2,C3

Source

Synthesized by IDT.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 284
  Illegal PstI site found at 864
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 284
  Illegal PstI site found at 864
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 106
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 284
  Illegal PstI site found at 864
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 284
  Illegal PstI site found at 864
  Illegal NgoMIV site found at 364
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 560
  Illegal SapI site found at 815
  Illegal SapI.rc site found at 17
  

References

• Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.