Part:BBa_J18903:Design
pSB1AT3F construction plasmid for protein fusion BioBricks
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 3434
Illegal suffix found in sequence at 10 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 3434
Illegal suffix found in sequence at 11 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 3434
Illegal suffix found in sequence at 1 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2473
Design Notes
This plasmid was constructed by PCR and InFusion recombination. 1) The pSB1AT3 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) Three 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.
See also:
Source
constructed from pSB1AT3