1 Registry Star
Part:BBa_K152009:Design
Zea mays Actin1 Intron RNAi Production Tool
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 14
Illegal XbaI site found at 23
Illegal SpeI site found at 154
Illegal PstI site found at 218 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 14
Illegal SpeI site found at 154
Illegal PstI site found at 218
Illegal NotI site found at 162 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 14
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 14
Illegal XbaI site found at 23
Illegal SpeI site found at 154
Illegal PstI site found at 218 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 14
Illegal XbaI site found at 23
Illegal SpeI site found at 154
Illegal PstI site found at 218 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 112
Design Notes
In order to utilize existing biobricks to make RNAi constructs, it was necessary to re-organize restriction site placement. The part is housed in the Biobrick plasmid pSB1A2 which was cut with EcoRI and PstI, and ligated to the MfeI and PstI cut RNAi tool. The PstI site was conserved, but MfeI and EcoRI are non-conservative compatible ends and so the plasmid's EcoRI site was killed allowing the construct's intrenal EcoRI site to be unique. The construct was originally planned to have this structure:
NdeI - EcoRI - XbaI - Intron - NotI - SpeI - Terminator - PstI
In fact, due to a last minute primer design error, what we got is:
NdeI - EcoRI - XbaI - Intron - SpeI - NotI -Terminator - PstI
This rather profoundly changes the cloning strategy one must use (resulting in no SpeI sites and two XbaI rather than one of each) but should not affect its practicality or function very much.
Source
Template free PCR using two complimentary primers donated by IDT: one 60 bp long, and the other 200 bp long.