Composite

Part:BBa_K4410005:Design

Designed by: Ruyi Shi   Group: iGEM22_Worldshaper-HZBIOX   (2022-09-19)


H1-argR-H2


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1033
    Illegal PstI site found at 335
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 1033
    Illegal PstI site found at 335
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1033
    Illegal PstI site found at 335
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1033
    Illegal PstI site found at 335
    Illegal NgoMIV site found at 279
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Through PCR, we extracted H1-argR-H2 from EcN, and combined H1-argR-H2 with pMD19 T Vector by TA cloning to construct Recombinant pMD19T. Then, we deleted argR gene to obtain pMD19-T linear plasmid with 1000bp homologous fragments upstream and downstream of argR by reverse PCR. At the same time, we obtained KanR gene with upstream and downstream 1000bp fragments by PCR. After that, we combined KanR gene and pMD19-T linear plasmid through one-step cloning to obtain recombinant pMD19 T in DH-5α bacteria colony, and then got Donor DNA. finally, we transformed donor DNA into EcN1917.


Source

This part is based on BBa_K4410000, BBa_K4410002 and BBa_K4410001.

References