HPP New Start and Finish

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July 23, 2011 New Oligos to Order START_TOP (67 nt)

AATTCGGTCTCAGACGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGTGGAGAGACGCTGCA

START_Bot (59 nt)

GCGTCTCTCCACGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAACGTCTGAGACCG

FINISH_Top (40 nt)

AATTCCGTCTCAGTGGGCATCAGTCGAGAGAGACCCTGCA

FINISH_Bot (32 nt)

GGGTCTCTCTCGACTGATGCCCACTGAGACGG NewHPPVec_Top (36 nt) AATTCGTCCAGAGACCGCTAGCGGTCTCACGAGCTGCA

NewHPPVec_Bot (32 nt) GCTCGTGAGACCGCTAGCGGTCTCTGGACG

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
                  /                                        /

BsaI

BsmBI

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. 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
             /                                     /
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