Difference between revisions of "Part:BBa K1352004:Design"
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===Design Notes=== | ===Design Notes=== | ||
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+ | 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. | ||
Revision as of 12:27, 4 October 2014
Ice Nucleation Protein (INP) Yellow Florescent Protein (YFP) FLAG-tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2324
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1036
- 1000COMPATIBLE 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