Difference between revisions of "HPP New Start and Finish"

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# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
 
# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
  
RI______BsaI>________START____________J23100___________Half_____<BsmBI___PstI<br>
+
  RI  BsaI>   START              J23100              Half  <BsmBI  PstI<br>
AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA<br>
+
AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA<br>
___G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G
+
    G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G
 
                  
 
                  
 
<br><br>
 
<br><br>
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# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
 
# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
  
RI   BsmBI>  /Half  Spacer  /FINISH  <BsaI  PstI<br>
+
  RI BsmBI>   Half   Spacer  FINISH  <BsaI  PstI<br>
AATTC CGTCTC A  GTGG  GCATCAGT  CGAG  A GAGACC CTGCA<br>
+
AATTC CGTCTC A  GTGG  GCATCAGT  CGAG  A GAGACC CTGCA<br>
G GCAGAG T  CACC  CGTAGTCA  GCTC  T CTCTGG G
+
    G GCAGAG T  CACC  CGTAGTCA  GCTC  T CTCTGG G
<br>
+
<br><br>
 
'''New HPP Vector'''
 
'''New HPP Vector'''
 
# 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).
 
# 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).
 
# NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
 
# NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
# Flanked by EcoRI and PstI sticky ends.
+
# Flanked by EcoRI and PstI sticky ends.<br><br>
  
'''Specific Words Used So Far'''
+
  RI    START  <BsaI  NheI  BsaI>  FINISH PstI<br>
START GTCC  GGAC
+
AATTC  GTCC  A GAGACC GCTAGC GGTCTC A  CGAG  CTGCA<br>
FINISH CGAG  CTCG
+
    G  CAGG  T CTCTGG CGATCG CCAGAG T  GCTC  G<br><br>
Half-Edge GTGG  CCAC
+
 
GFP GGGT    ACCC
+
'''Specific Words Used So Far'''<br><br>
RFP CTGC  GCAG
+
START GTCC  GGAC <br>
LacZ CCTG  CAGG
+
FINISH CGAG  CTCG<br>
AspC CGTC  GACG
+
Half-Edge GTGG  CCAC<br>
PyrE GGCG  CGCC
+
GFP GGGT    ACCC<br>
ilvE GGGG  CCCC
+
RFP CTGC  GCAG<br>
CAT CGGT  ACCG
+
LacZ CCTG  CAGG<br>
 +
AspC CGTC  GACG<br>
 +
PyrE GGCG  CGCC<br>
 +
ilvE GGGG  CCCC<br>
 +
CAT CGGT  ACCG<br>
 
TyrB CGCA  TGCG<br>
 
TyrB CGCA  TGCG<br>
  
RI  START    <BsaI    NheI    BsaI>    FINISH  PstI
+
 
AATTC  GTCC  A GAGACC GCTAGC GGTCTC A  CGAG  CTGCA<br>
+
Diagram of HPP vector Design: [[Media:MOWestidea.pptx]]
G  CAGG  T CTCTGG CGATCG CCAGAG T  GCTC  G<br>
+
<br>

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