Difference between revisions of "HPP New Start and Finish"
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'''New START Half Edge''' | '''New START Half Edge''' | ||
| − | + | # Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector. | |
| − | + | # Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge. | |
| − | + | # In between these is a J23100 promoter. | |
| − | + | # Flanked by EcoRI and PstI sticky ends for cloning.<br><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 | |
<br><br> | <br><br> | ||
'''New FINISH Half Edge''' | '''New FINISH Half Edge''' | ||
| − | + | # Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge. | |
| − | + | # Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector. | |
| − | + | # In between these is a 8 nt spacer. | |
| − | + | # Flanked by EcoRI and PstI sticky ends for cloning.<br><br> | |
| − | RI | + | 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). | |
| − | + | # 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.<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> | ||
| − | + | ||
| − | + | Diagram of HPP vector Design: [[Media:MOWestidea.pptx]] | |
| − | + | <br> | |
Latest revision as of 20:27, 26 August 2011
New START Half Edge
- Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector.
- Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge.
- In between these is a J23100 promoter.
- 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
- Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge.
- Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector.
- In between these is a 8 nt spacer.
- 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
- 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.
- 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
