Coding

Part:BBa_K1352004:Design

Designed by: James Long   Group: iGEM14_Aberdeen_Scotland   (2014-10-03)
Revision as of 14:44, 4 October 2014 by JamesLongAberdeen (Talk | contribs) (→‎Design Notes)

Ice Nucleation Protein (INP) Yellow Florescent Protein (YFP) FLAG-tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2324
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1036
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Creation of INP-YFP-FLAG fragments followed by InFusion cloning Four infusion primers (primers with one homologous half (lower case) to the template, and one “overhang” half (upper case), the last 15 nucleotides of which is homologous to another DNA fragment) were designed.

“INP-VEC-F” (CGCGGCCGCTTCTAGAttaatacgactcactataggg)

“INP-FLAG-R” (AAGCTTTTTATCATCATCATCTTTATAATCAGATCTcttgtacagctcgtccatgc)

“INP-FLAG-F” (AGATCTGATTATAAAGATGATGATGATAAAAAGCTTtaataatactagcaacatatcataacggagtg)

“INP-VEC-R” (AGCGGCCGCTACTAGTtataaacgcagaaaggccc)

Two INP-YFP-FLAG fragments were created by PCR amplification, both use K523013 as the template, the first uses “INP-VEC-F” and “INP-FLAG-R” primers, the second uses “INP-VEC-R” and “INP-FLAG-F” primers. The Clontech InFusion kit was used to recombine the two INP-YFP-FLAG fragments with pSB1C3 backbone (cut with Xba1 and Spe1); this kit was followed according to the manufacturer’s instructions.

Source

This part was adapted from BBa_K523013

References