Difference between revisions of "Part:BBa K1638006:Design"

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===Design Notes===
 
===Design Notes===
−
Standard assembly [RFC10] with this part creates scarsite (uacuag). Scarsites encodes stopcodons, which is inconvenient for the right use of this linker. The linker instead contains a 5'-end BamHI restriction site to be used in fusion to proteins with a 3'-end BamHI restriction site.  For implementation, we recommend the following assembly method:
+
Standard assembly RFC[10] with this part creates scarsite (uacuag). Scarsites encodes stopcodons, which is inconvenient for the right use of this linker. The linker instead contains a 5'-end BamHI restriction site to be used in fusion to proteins with a 3'-end BamHI restriction site.  For implementation, we recommend the following assembly method:
  
 
1) Design primers for amplification of the C-terminal fusion protein with the DNA sequence for the linker in the overhang of the forward primer.
 
1) Design primers for amplification of the C-terminal fusion protein with the DNA sequence for the linker in the overhang of the forward primer.
 +
 
1.2) Forward primer: 5'-CGCTTCTAGAGGGATCCGAAAATTTGTATTTTCAATCTGGTNNN...NNN-3' - (XbaI res. site and linker(with BamHI res. site) included)
 
1.2) Forward primer: 5'-CGCTTCTAGAGGGATCCGAAAATTTGTATTTTCAATCTGGTNNN...NNN-3' - (XbaI res. site and linker(with BamHI res. site) included)
 +
 
1.3) Reverse primer:  5'-ATATCTGCAGCGGCCGCTACTAGTANNN...NNN-3' (SpeI and PstI res. site included)
 
1.3) Reverse primer:  5'-ATATCTGCAGCGGCCGCTACTAGTANNN...NNN-3' (SpeI and PstI res. site included)
 +
 
2) Run PCR with  designed primers.
 
2) Run PCR with  designed primers.
 +
 
3) Digest PCR-products and a standard backbone (e.g. pSB1C3) with XbaI and PstI
 
3) Digest PCR-products and a standard backbone (e.g. pSB1C3) with XbaI and PstI
 +
 
4) Ligate backbone and PCR product
 
4) Ligate backbone and PCR product
  
  
−
Addition of linker_C-terminalFusionProtein to N-terminal fusion protein:
+
'''Addition of linker_C-terminalFusionProtein to N-terminal fusion protein:'''
 +
 
 
5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site.
 
5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site.
−
5.1) Forward: 5'-CGCTTCTAGAGNNN...NNN-3' (includes XbaI res. site)
+
 
 +
5.1) Forward: 5'-CGTCTGGAATTCGCGGCCGCTTCTAGAGNNN...NNN-3' (includes EcoRI and XbaI res. site)
 +
 
 
5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)
 
5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)
 +
 
6) Run PCR with designed primers.
 
6) Run PCR with designed primers.
−
7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with BamHI and PstI(or SpeI) res. site.
+
 
−
8) Ligate backbone PCR product
+
7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with EcoRI and BamHI.
 +
 
 +
8) Ligate backbone and PCR product
  
 
===Source===
 
===Source===

Latest revision as of 15:56, 14 August 2015


10 aa linker with BamHI restriction site and TEV recognition site


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Standard assembly RFC[10] with this part creates scarsite (uacuag). Scarsites encodes stopcodons, which is inconvenient for the right use of this linker. The linker instead contains a 5'-end BamHI restriction site to be used in fusion to proteins with a 3'-end BamHI restriction site. For implementation, we recommend the following assembly method:

1) Design primers for amplification of the C-terminal fusion protein with the DNA sequence for the linker in the overhang of the forward primer.

1.2) Forward primer: 5'-CGCTTCTAGAGGGATCCGAAAATTTGTATTTTCAATCTGGTNNN...NNN-3' - (XbaI res. site and linker(with BamHI res. site) included)

1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN...NNN-3' (SpeI and PstI res. site included)

2) Run PCR with designed primers.

3) Digest PCR-products and a standard backbone (e.g. pSB1C3) with XbaI and PstI

4) Ligate backbone and PCR product


Addition of linker_C-terminalFusionProtein to N-terminal fusion protein:

5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site.

5.1) Forward: 5'-CGTCTGGAATTCGCGGCCGCTTCTAGAGNNN...NNN-3' (includes EcoRI and XbaI res. site)

5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)

6) Run PCR with designed primers.

7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with EcoRI and BamHI.

8) Ligate backbone and PCR product

Source

..

References