Difference between revisions of "Part:BBa K3505023"
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===Usage and Biology===
 
===Usage and Biology===
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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).
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===Design Notes===
 
===Design Notes===
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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.  
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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 GoldenBraid cloning.  
 
The CDS has position B2-B5.
 
The CDS has position B2-B5.
  
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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     [[Image: T--Thessaly--GB-CCAT-GCTT.jpeg|900px|thumb|none|<i><b>Figure 1.</b>The overhangs of this part in the Golden Braid Grammar.</i>]]
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     [[Image: T--Thessaly--GB-CCAT-GCTT.jpeg|900px|thumb|none|<i><b>Figure 1.</b>The overhangs of this part in the GoldenBraid Grammar.</i>]]
  
 
   [[Image:T--Thessaly--FFAR2-snap.png|900px|thumb|none|<I><b>Figure 2.</b> The level 0 module : pupd2-FFAR2:V2tail:TCS (illustration from SnapGene)</i>]]
 
   [[Image:T--Thessaly--FFAR2-snap.png|900px|thumb|none|<I><b>Figure 2.</b> The level 0 module : pupd2-FFAR2:V2tail:TCS (illustration from SnapGene)</i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--FFAR2-ECFP-digestion.png|700px|thumb|none|<i><b>Fig.1:</b>(U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204 bp, Positive Result:C2,C3</i>]]
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[[File:T--Thessaly--FFAR2-ECFP-digestion.png|700px|thumb|none|<i><b>Fig.3:</b>(U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204 bp, Positive Result:C2,C3</i>]]
  
 
===Source===
 
===Source===
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3505023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3505023 SequenceAndFeatures</partinfo>
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===References===
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*Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.

Latest revision as of 00:28, 28 October 2020


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 GoldenBraid cloning. The CDS has position B2-B5.

Figure 1.The overhangs of this part in the GoldenBraid Grammar.
Figure 2. The level 0 module : pupd2-FFAR2:V2tail:TCS (illustration from SnapGene)

Verification of Cloning

Fig.3:(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

  • Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.