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)
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PCR gh1000F/gh1000R on A+B+C           (622 bp, EcoRI/BamHI)
Digest pBjk2741                     (EcoRI/BamHI, 2170+910 L)
Product is pBjk2741-<FokI-!
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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 <FokI-!       gcaaaGGATCCTTAaaaattgatctcgccattgttg

Construction of sbb35: P9 rep Protein - {rbs.ColE2 RepA}
Already exists just need to do a EcoR1/BamH1 transfer

JCA Notes

• 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

 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

Part:  GATCTCAGCTGGTTaaatctgaactggaggagaaaaaatccgagctgcgccacaaactgaaatatgtgcctcacgagtatatcgaactgatcgagatcgcccgtaatagtacccaagaccgtatcctggaaatgaaagtgatggagttcttcatgaaagtctatggctatcgtggcaaacatctgggtggtagccGTAAACCAGACGGTGCCATTTATACcgttggttccccgatcgattatggcgttatcgtggAcacaaaagcgtattctggcggttataatctgccgattggtcaggctgatgagatggaacgttatgtggaagagaatcagacccgtaacaaacatctgaacccgaacgaatggtggaaagtgtatccgtcaagtgtcaccgagttcaaatttctgttcgtgagcggccactttaaaggcaactataaagcccagctgactcgtctgaaccatatcaccaatTgcaatggggcagtgctgagtgttgaggaactgctgatcggtggagaaatgatcaaagcaggcaccctgactctggaagaagttcgccgtaaattCAACAATGGCGAGATCAATTTTTAAG