Part:BBa_J18905:Design

pSB1AK3X Freiburg Fusion -> BioFusion conversion plasmid

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3173
  Illegal suffix found in sequence at 6
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3173
  Illegal SpeI site found at 7
  Illegal PstI site found at 21
  Illegal NotI site found at 14
  Illegal NotI site found at 3179
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3173
  Illegal XhoI site found at 1040
  Illegal XhoI site found at 2066
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3173
  Illegal suffix found in sequence at 7
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3173
  Illegal XbaI site found at 3188
  Illegal SpeI site found at 7
  Illegal PstI site found at 21
  Illegal NgoMIV site found at 3194
  Illegal AgeI site found at 1
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2212
  

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.

All 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 pSB1AK3. Only insert and flanks have been verified by sequencing.

Source

constructed from pSB1AK3