Difference between revisions of "Part:BBa K4225013:Design"

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===Design Notes===
 
===Design Notes===
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<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
  
 
===Source===
 
===Source===
  
 
===References===
 
===References===

Revision as of 15:22, 12 October 2022


Inhibitory Sequence (IS)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes


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

Source

References