Part:BBa_K3407003:Design
YmdB: a regulator of RNAseIII activity in E. coli.
- 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
The gene was amplified by PCR from E.coli Bl21(DE3) genome as a template. YmdB amino acid sequence was taken from Uniprot (Uniprot ID: P0A8D6), it was introduced as a query in tBlastn from NCBI, against Escherichia coli BL21(DE3) genome and the resulting YmdB gene sequence (GenBank: AM946981.2) 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 Assembly. The primers used were the following:
M3-037 (YmdB - Fw):
5’ - gaattcaaaagatcttttaagaaggagatatacatATGAAAACGCGTATTCATGTTGTGC - 3’
M3-038 (YmdB - Rv):
5’- tttatttgatgcctggagatccttactcgagtttggatccttaGTGATGGTGGTGATGATGGTGGTGATGGTGACCTGTACTTCCTGTTTCATCTCCTTGTTGGGTAAGGAGTC - 3’
Source
Plasmid used pBbA2k backbone from BglBricks. pBbA2k-RFP was a gift from Jay Keasling (Addgene plasmid # 35327 ; http://n2t.net/addgene:35327 ; RRID:Addgene_35327) [1]. Please refer to the Addgene page for more information about licences associated with the use of the plasmid.
References