Part:BBa_K3407018:Design
Mini-3 endoribonuclease with araBAD promoter - RBS - and rrnBT1 terminator
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 300
Illegal XbaI site found at 673
Illegal PstI site found at 527 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 300
Illegal PstI site found at 527 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 300
Illegal BglII site found at 309
Illegal BamHI site found at 809
Illegal XhoI site found at 818 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 300
Illegal XbaI site found at 673
Illegal PstI site found at 527 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 300
Illegal XbaI site found at 673
Illegal PstI site found at 527
Illegal AgeI site found at 74 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 56
Design Notes
The gene was amplified by PCR from Bacillus subtilis strain 168, which has the ATCC6051 genome that is the same strain as NCCB 70064, also same as KTCC 1028. Mini-3 amino acid sequence was retrieved from UniProt (UniProt ID: O31418), introduced as a query in tBlastn from NCBI against B. subtilis ATCC 6051 genome and the resulting mini3 sequence (GenBank: CP011115.1) was used as a template for the design of the primers. Primers were designed to have a melting temperature of 59-60 ºC. Overhangs with BglBricks Prefix and Suffix were added, along with a His-tag in the Reverse primer for a C-terminal-tagged protein. Designed to be cloned through Gibson Assembly. The primers used were the following:
M3-039 (mini3-Fw)
5’ - gaattcaaaagatcttttaagaaggagatatacatATGCTTGAATTTGATACGATAAAAGATTCTAAGCAG - 3’
M3-040 (mini3-Rv)
5’ - tttatttgatgcctggagatccttactcgagtttggatccTTAGTGGTGGTGGTGATGGTGGTCGCTTTTTACGCTGGTTTCCTCTGTTGCTGACTCATTTGTTTTCCTCC - 3’
Source
Genomic DNA from B. subtilis strain 168. Plasmid used pBbE8c backbone from BglBricks. pBbE8c-RFP was a gift from Jay Keasling (Addgene plasmid # 35269; http://n2t.net/addgene:35269; RRID:Addgene_35269) [1] Please refer to the Addgene page for more information about licences associated with the use of the plasmid.
References