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)(Dogra, Sona, Kumar and Yadav, 2016). 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