Part:BBa_J18906:Design
pSB1AT3X Freiburg Fusion -> BioFusion conversion plasmid
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 3430
Illegal suffix found in sequence at 6 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3430
Illegal NheI site found at 1275
Illegal SpeI site found at 7
Illegal PstI site found at 21
Illegal NotI site found at 14
Illegal NotI site found at 3436 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3430
Illegal BamHI site found at 1421
Illegal XhoI site found at 1040
Illegal XhoI site found at 2323 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 3430
Illegal suffix found in sequence at 7 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 3430
Illegal XbaI site found at 3445
Illegal SpeI site found at 7
Illegal PstI site found at 21
Illegal NgoMIV site found at 3451
Illegal AgeI site found at 1 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2469
Design Notes
This plasmid was constructed by PCR and InFusion recombination.
1) Three NgoMIV restriction sites within the TetR gene of pSB1AT3 were removed using the QuickChange kit (Qiagen)
2) The pSB1AT3 vector backbone was linearized by PCR, introducing the NgoMIV / AgeI restriction sites
3) The P1010 insert was amplified by PCR, introducing the NgoMIV / AgeI restriction sites
4) The two overlapping PCR products were recombined using the clonetech InFusion kit.
All PCR reactions (except the QickChange one) 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.
Source
constructed from pSB1AT3