Part:BBa_K3505024

B-arrestin:TEV Protease GB compatible with B2-B5

Usage And Biology

B-arrestin has the ability to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease.TEV protease recognizes and cleaves the cleavage site. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (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 GoldenBraid Grammar.

Figure 2. The level 0 module : pupd2-b-arrestin-TEV protease (illustration from SnapGene)

Verification of Cloning

Fig.3:(U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1611
  Illegal PstI site found at 303
  Illegal PstI site found at 390
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1611
  Illegal PstI site found at 303
  Illegal PstI site found at 390
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 203
  Illegal XhoI site found at 600
  Illegal XhoI site found at 994
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1611
  Illegal PstI site found at 303
  Illegal PstI site found at 390
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1611
  Illegal PstI site found at 303
  Illegal PstI site found at 390
  Illegal AgeI site found at 586
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1545
  Illegal SapI.rc site found at 1893