Difference between revisions of "Part:BBa J18903:Design"

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===Design Notes===
 
===Design Notes===
 
This plasmid was constructed by PCR and InFusion recombination.  
 
This plasmid was constructed by PCR and InFusion recombination.  
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1) The pSB1AK3 vector backbone was linearized by PCR, introducing the NgoMIV / AgeI restriction sites
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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
 
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.
 
3) The two overlapping PCR products were recombined using the Clonetech InFusion kit.
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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.
 
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.
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 +
See also:
 +
 +
* [https://parts.igem.org/Part:BBa_J18901 pSB1AC3F]
 +
 +
* [https://parts.igem.org/Part:BBa_J18902 pSB1AK3F]
  
 
===Source===
 
===Source===

Latest revision as of 13:29, 11 August 2008

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 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

References