Part:BBa_J18903:Design

pSB1AT3F construction plasmid for protein fusion BioBricks

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3434
  Illegal suffix found in sequence at 10
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3434
  Illegal NheI site found at 1279
  Illegal SpeI site found at 11
  Illegal PstI site found at 25
  Illegal NotI site found at 18
  Illegal NotI site found at 3440
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3434
  Illegal BamHI site found at 1425
  Illegal XhoI site found at 1044
  Illegal XhoI site found at 2327
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3434
  Illegal suffix found in sequence at 11
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3434
  Illegal suffix found in sequence at 1
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2473
  

Design Notes

This plasmid was constructed by PCR and InFusion recombination. 1) The pSB1AK3 vector backbone was linearized by PCR, introducing the NgoMIV / AgeI restriction sites 2) The P1010 insert was amplified by PCR, introducing the NgoMIV / AgeI restriction sites 3) The two overlapping PCR products were recombined using the Clonetech InFusion kit. 4) Two NgoMIV restriction sites in the original pSB1AT3 (within the TcR) were deleted following the QuickChange protocol (from Qiagen).

PCR reactions were performed with a proof-reading polymerase featuring a very low mutation rate. Nevertheless, the vector backbone may contain mutations with respect to pSB1AT3. Only insert and flanks have been verified by sequencing. The deletion of NgoMIV sites has been verified by restriction digests.

Source

constructed from pSB1AT3