Difference between revisions of "Part:BBa K5034219:Design"
Line 8: Line 8:
 
===Design Notes===
 
===Design Notes===
  
−
PAP gene was PCR-amplified and inserted into the plasmid pYYDT(after NdeI and XhoI digestion) to get the expression vector. It then got transformed into <i>E. coli</i> DH5&#945; to amplify, and was purified by picking monoclonal culture and sequencing.
+
PAP gene was PCR-amplified and inserted into the plasmid pBBR1MCS-terminator(after EcoRI and XhoI digestion) to get the expression vector. It then got transformed into <i>E. coli</i> DH5&#945; to amplify, and was purified by picking monoclonal culture and sequencing.
  
−
The Terminator(BBa_B0015) is on the plasmid backbone(BBa_K5034201).
+
The terminator(BBa_B0015) is on the plasmid backbone(BBa_K5034201).
  
 
===Source===
 
===Source===

Revision as of 11:23, 30 September 2024


Poly P + AMP-> ADP


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4981
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2834
    Illegal NotI site found at 4987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4981
    Illegal BglII site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4981
    Illegal XbaI site found at 4996
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 562
    Illegal NgoMIV site found at 4244
    Illegal NgoMIV site found at 4527
    Illegal AgeI site found at 402
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

PAP gene was PCR-amplified and inserted into the plasmid pBBR1MCS-terminator(after EcoRI and XhoI digestion) to get the expression vector. It then got transformed into E. coli DH5α to amplify, and was purified by picking monoclonal culture and sequencing.

The terminator(BBa_B0015) is on the plasmid backbone(BBa_K5034201).

Source

Phosphate-AMP phosphotransferase(PAP) from Acinetobacter johnsonii. NCBI reference sequence: AB092983.1

References

Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181. doi:10.1128/jb.186.15.5178-5181.2004