Difference between revisions of "Part:BBa K2591013:Design"
(→Source) |
(→References) |
||
Line 15: | Line 15: | ||
===References=== | ===References=== | ||
+ | |||
+ | Jackson EL; Willis N; Mercer K; Bronson RT; Crowley D; Montoya R; Jacks T; Tuveson DA. 2001. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15(24):3243-8PubMed: 11751630MGI: J:73445 |
Revision as of 07:18, 7 October 2018
LoxP-BGH polyA-LoxP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
First, we use primers suffix_F(5'-TACTAGTAGCGGCCGCTGCAG-3') and prefix_R(5'-CTCTAGAAGCGGCCGCGAATTC-3') to do a reverse PCR with pSB1C3 backbone, obtaining the linearized backbone. Then we design the primers Prefix_LoxP_bGHpA_F(5'-GAATTCGCGGCCGCTTCTAGAGATAACTTCGTATAGCATACATTATACGAAGTTATTAGAGCTCGCTGATCAGCCTC-3') and Suffix_LoxP_bGHpA_R(5'-CTGCAGCGGCCGCTACTAGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCTCTCCCCAGCATGCCTGCTATTcTC-3') containing LoxP sequence, also, prefix and suffix sequence are added. Using these two primers and BGH polyA containing template to do PCR, we got the LSL part and then used Gibson Assembly to complete the plasmid.
Source
LoxP sequence is from synthesis, BGH polyA is from our host lab.
References
Jackson EL; Willis N; Mercer K; Bronson RT; Crowley D; Montoya R; Jacks T; Tuveson DA. 2001. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15(24):3243-8PubMed: 11751630MGI: J:73445