Project
Part:BBa_K404252:Design
Designed by: Freiburg Bioware 2010 Group: iGEM10_Freiburg_Bioware (2010-10-14)
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-Empty)
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2396
Illegal XhoI site found at 698
Illegal XhoI site found at 884 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 665
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2922
Illegal SapI site found at 1833
Design Notes
Production of ViralBricks
The ViralBricks provided in Freiburg Bioware's Virus Construction Kit were predominantly ordered as oligonucleotides. These oligos were hybridized and subsequently cloned into the pSB1C3-001_ViralBrick-587-Beta Lactamase which functions as the multiple cloning site for the ViralBricks.
ViralBricks with the motif insertion Z34C(length of up to 189bp) were orderd as oligos with an overlap of 30 nucleotides. These pairs of oligonucleotides were hybridized, filled up with the Klenow-fragment and cloned into the ViralBrick MCS.
The ViralBricks 453-Empty and 587-Empty could be cloned from an ordered gene synthese directly into the ViralBrick MCS.
Loop insertions employing ViralBricks is not based on RFC restriction sites but on the four restriction sites SspI, SalI, BamHI and PvuII. These restriction endonucleases were selected because they were below average present in the capsid coding constructs of the AAV-2 and could be included near the insertion sites at position 453 & 587 in an silent manner. For this purpose we introduced two pointmutations in pSB1C3 to clean the standard backbone pSB1C3 from these restriction sites. All ViralBricks were designed in an way that the sequences that are ment to replace the loop region are flanked by RFC25 restriction sites in order to be able to submit it to the Parts Registry. These additional restriction sites allow to modify fully assembled composite parts by including functional motifs into loop coding sequences in an single cloning step.
Loop insertions employing ViralBricks is not based on RFC restriction sites but on the four restriction sites SspI, SalI, BamHI and PvuII. These restriction endonucleases were selected because they were below average present in the capsid coding constructs of the AAV-2 and could be included near the insertion sites at position 453 & 587 in an silent manner. For this purpose we introduced two pointmutations in pSB1C3 to clean the standard backbone pSB1C3 from these restriction sites. All ViralBricks were designed in an way that the sequences that are ment to replace the loop region are flanked by RFC25 restriction sites in order to be able to submit it to the Parts Registry. These additional restriction sites allow to modify fully assembled composite parts by including functional motifs into loop coding sequences in an single cloning step.
Source
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