Part:BBa_K2496007
DPP4(weak)
DPP4(weak) Weak DPP4 is consist of three part. Anderson promoter(BBa_J23119)+FLAG(BBa_J2496000)+DPP4(BBa_J2496000). Expression of DPP4 is regulated by the Anderson promoter. iGEM initially encouraged the teams to identify the presence of EcoR1 or Pst1 site. The DPP4 sequence contains two Pst1 sites. Accordingly, the additional presence of the particular site that restriction enzyme is likely to be mistakenly recognized, leading to decomposition of DPP4. Ligation process of infusion cloning method dose not require restriction enzyme cutting of insert gene. So, we can utilize the gene we want even if there are EcoR1, Pst1 sites. Sequence of Anderson promoter is extraordinarily Short=33bp. Thus, it becomes comparatively inefficient to check the ligation. We added Anderson promoter to PCR primer sequence by using primer design method for infusion ligation process, which ensures accuracy and convenience. The success of ligation can be manifested by the success of PCR. Although 33bp is so short that it cannot be precisely detected by gel band, proper indication on the band can confirm the cusses of gene ligation of Anderson promoter. In order to add DPP4 to the final vector pSB1C3, the vector pCDNA3 of the process in between should have DPP4 first. It is later sued as a template of PCR for the insert gene. How we designed the primer for infusion cloning For primer : vector seq(pSB1C3) + enzyme(EcoR1)+Anderson seq(weak)+flag primer seq Re primer : vector seq(pSB1C3) + enzyme seq(Pst1)+DPP4seq
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 791
Illegal PstI site found at 1565 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1781
Illegal NheI site found at 2282
Illegal PstI site found at 791
Illegal PstI site found at 1565 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2208
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 791
Illegal PstI site found at 1565 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 791
Illegal PstI site found at 1565 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 702
Illegal BsaI site found at 2069
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