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Cut Site Design
From 2011.igem.org
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omega..GCCAtGAGACC...cutout...GGTCTCaACGG..F2 | omega..GCCAtGAGACC...cutout...GGTCTCaACGG..F2 | ||
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Latest revision as of 15:50, 3 August 2011
Cut Site Design
General BsaI cut sites
GGTCTCN
CCAGAGNNNNN
NNNNNNGAGACC
NCTCTGG
Flanking Amino Acids
upstream, last two AA of omega: TP (NCCN) (we used GCCA) downstream, first two AA of finger 2: TG (ACNG) (we used ACGG)
Our BsaI cut sites:
GCCA at 5' end ACGG at 3' end
Set N to A on the top strand of the recognition site. Also, make it lowercase in sequences for easy reference.
Our Schematic:
plasmid oligo GGTCTa---- GCCAtGAGACC CCAGAtCGGT and ----aCTCTGG for 5' oligo plasmid GGTCTa---- ACGGtGAGACC CCAGAtTGCC and ----aCTCTGG for 3'
Oligo:
|| ||
F_primer..GGTCTCAgcca....ZF.......ACGGTGAGACC..R_primer
F_primer..CCAGAGTCGGT....ZF.......tgccACTCTGG..R_primer
|| ||
gcca .... ----
---- .... tgcc
Plasmid:
omega || cutout || F2
omega..GCCATGAGACC...cutout...GGTCTCAacgg....
omega..cggtACTCTGG...cutout...CCAGAGTTGCC..F2
|| ||
---- ..... acgg
cggt ..... ----
Final Oligo Tag Flanks:
F_primer..GGTCTCaGCCA...ZF...ACGGtGAGACC..R_primer
Final Plasmid Cut Insert:
omega..GCCAtGAGACC...cutout...GGTCTCaACGG..F2
