Template:SBB10 ConstructionFiles LuoJ sbb20

Construction of sbb20: FokI Cleavage Domain - <FokI-! PCR gh1000F/gh1001R on BBa_K157000  (261 bp, gp = A) PCR gh1001F/gh1003R on BBa_K157000   (264 bp, gp = B) PCR gh1003F/gh1000R on BBa_K157000   (144 bp, gp = C) --- PCR gh1000F/gh1000R on A+B+C          (622 bp, EcoRI/BamHI) Digest pBjk2741                    (EcoRI/BamHI, 2170+910 L) Product is pBjk2741-<FokI-! --- gh1000F Forward for part <FokI-! ccagtGAATTCatgAGATCTCAGCTGGTTaaatctgaactggaggagaaaaaatccg gh1001F Making internal mutation 1+2 cgttggttccccgatcgattatggcgttatcgtggAcacaaaagc gh1001R Making internal mutation 1+2 cgccataatcgatcggggaaccaacggtataaatggcaccGTctggtttac gh1003F Making internal mutation 3   ccatatcaccaatTgcaatgg gh1003R Making internal mutation 3   ccattgcAattggtgatatgg gh1000R Reverse for part 

JCA Notes
Construction of sbb20: FokI Cleavage Domain - <FokI-! PCR gh1000F/gh1001R on BBa_K243001  (261 bp, gp = A) PCR gh1001F/gh1003R on BBa_K243001   (264 bp, gp = B) PCR gh1003F/gh1000R on BBa_K243001   (144 bp, gp = C) --- PCR gh1000F/gh1000R on A+B+C          (622 bp, EcoRI/BamHI) Digest pBjk2741                    (EcoRI/BamHI, 2170+910 L) Product is pBjk2741-sbb20 --- gh1000F Forward for part <FokI-! ccagtGAATTCatgAGATCTCAGCTGGTTaaatctgaactggaggag gh1001F Making internal mutation 1+2 cgttggttccccgatcgattatggcgttatcgtggAcacaaaagc gh1001R Making internal mutation 1+2 cgccataatcgatcggggaaccaacggtataaatggcaccGTctggtttac gh1003F Making internal mutation 3   ccatatcaccaatTgcaatggggcagtgctgag gh1003R Making internal mutation 3   ccattgcAattggtgatatgg gh1000R Reverse for part <FokI-! gcaaaGGATCCTTAaaaattgatctcgccattgttg
 * Assembles correctly
 * Name of plasmid should be partName, not partDescription
 * First oligo would be better without the a's even though it's a shorter anealing region
 * I extended gh1003F, it looked a little iffy on the 3' end

Part: GATCTCAGCTGGTTaaatctgaactggaggagaaaaaatccgagctgcgccacaaactgaaatatgtgcctcacgagtatatcgaactgatcgagatcgcccgtaatagtacccaagaccgtatcctggaaatgaaagtgatggagttcttcatgaaagtctatggctatcgtggcaaacatctgggtggtagccGTAAACCAGACGGTGCCATTTATACcgttggttccccgatcgattatggcgttatcgtggAcacaaaagcgtattctggcggttataatctgccgattggtcaggctgatgagatggaacgttatgtggaagagaatcagacccgtaacaaacatctgaacccgaacgaatggtggaaagtgtatccgtcaagtgtcaccgagttcaaatttctgttcgtgagcggccactttaaaggcaactataaagcccagctgactcgtctgaaccatatcaccaatTgcaatggggcagtgctgagtgttgaggaactgctgatcggtggagaaatgatcaaagcaggcaccctgactctggaagaagttcgccgtaaattCAACAATGGCGAGATCAATTTTTAAG