Cut Site Design

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