Difference between revisions of "Part:BBa K4225019:Design"

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===Design Notes===
 
===Design Notes===
 
This composite part is codon optimized for our chassis, DH5α. Spacers are also added between the parts.
 
This composite part is codon optimized for our chassis, DH5α. Spacers are also added between the parts.
 +
<br>The inhibitory sequence comprises of a serine codon, SfoI cut site and 77 amino acids from the C-terminal of mRFP. The placement of serine downstream of the TEV protease cut site is necessary for its effective cleavage.
 +
<br>
 +
<br> This part was constructed by PCR of Ptac-TEV-Pc-GFP-LVA-CS (BBa_K4225018) with a forward primer (attaches to the prefix) and a reverse primer (attaches to the suffix) that has an overhang with a Sfol cut site. The PCR product is then digested with EcoRi and Sfol to form an intermediate insert(A).
 +
<br>
 +
<br>katGp-RFP(BBa_K4225005) that was used in another part of the project is also PCRed(with prefix and suffix primers, with no overhand) and cut with SfoI and PstI to form intermediate insert(B)
 +
<br>
 +
<br>The intermediate inserts A and B were then ligated with linearised pSB1C3 to form this plasmid

Latest revision as of 15:29, 12 October 2022


Ptac-TEV-Pc-GFP-LVA-CS-IS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1020
    Illegal NheI site found at 1043
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 855
    Illegal XhoI site found at 165
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1759
    Illegal SapI site found at 1902
    Illegal SapI.rc site found at 439
    Illegal SapI.rc site found at 787


Design Notes

This composite part is codon optimized for our chassis, DH5α. Spacers are also added between the parts.
The inhibitory sequence comprises of a serine codon, SfoI cut site and 77 amino acids from the C-terminal of mRFP. The placement of serine downstream of the TEV protease cut site is necessary for its effective cleavage.

This part was constructed by PCR of Ptac-TEV-Pc-GFP-LVA-CS (BBa_K4225018) with a forward primer (attaches to the prefix) and a reverse primer (attaches to the suffix) that has an overhang with a Sfol cut site. The PCR product is then digested with EcoRi and Sfol to form an intermediate insert(A).

katGp-RFP(BBa_K4225005) that was used in another part of the project is also PCRed(with prefix and suffix primers, with no overhand) and cut with SfoI and PstI to form intermediate insert(B)

The intermediate inserts A and B were then ligated with linearised pSB1C3 to form this plasmid