Difference between revisions of "HPP New Start and Finish"

Latest revision as of 20:27, 26 August 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.
   
   

 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

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

Diagram of HPP vector Design: Media:MOWestidea.pptx