Difference between revisions of "HPP New Start and Finish"

Revision as of 20:20, 27 July 2011

New START Half Edge

1. Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector.
2. Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge.
3. In between these is a J23100 promoter.
4. Flanked by EcoRI and PstI sticky ends for cloning.
   
   

RI______BsaI>________START____________J23100___________Half_____<BsmBI___PstI
AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA
___G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G

New FINISH Half Edge

1. Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge.
2. Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector.
3. In between these is a 8 nt spacer.
4. Flanked by EcoRI and PstI sticky ends for cloning.
   
   

RI BsmBI> /Half Spacer /FINISH <BsaI PstI
AATTC CGTCTC A GTGG GCATCAGT CGAG A GAGACC CTGCA
G GCAGAG T CACC CGTAGTCA GCTC T CTCTGG G
New HPP Vector

1. Use BsaI to free up a new START word and a new FINISH word that are different from any of the Gene words (listed below).
2. NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
3. Flanked by EcoRI and PstI sticky ends.

Specific Words Used So Far START GTCC GGAC FINISH CGAG CTCG Half-Edge GTGG CCAC GFP GGGT ACCC RFP CTGC GCAG LacZ CCTG CAGG AspC CGTC GACG PyrE GGCG CGCC ilvE GGGG CCCC CAT CGGT ACCG TyrB CGCA TGCG

RI START <BsaI NheI BsaI> FINISH PstI AATTC GTCC A GAGACC GCTAGC GGTCTC A CGAG CTGCA
G CAGG T CTCTGG CGATCG CCAGAG T GCTC G