Designed by: Shinjini Saha Group: iGEM14_MIT (2014-10-17)
pENTR_miRNAG1
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 502
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 502
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 502 Illegal XhoI site found at 398
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 502
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 502
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1024 Illegal SapI.rc site found at 16
This part was created using scarless golden gate assembly. This basic part is flanked by L1 and L2 sites and can be easily cloned into an entry vector using an LR reaction. A promoter can be easily inserted in front of this part in a one pot LR reaction with a promoter flanked by L4 and R1 sites cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts.
In our miRNA-generating vector, the sequence of the premature miRNA is contained in between exons coding for mKate2. If the miRNA is processed successfully, the premature miRNA-sequence will be spliced out of the vector, leaving just an intact mKate2 coding sequence in the vector. So red fluorescence via mKate2 is our proxy for determining effective processing of our miRNA’s.
