Difference between revisions of "Part:BBa K3407019:Design"
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===Design Notes=== | ===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=== | ===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) <html><a href="#1">[1]</a></html>. | |
+ | Please refer to the Addgene page for more information about licences associated with the use of the plasmid. | ||
===References=== | ===References=== | ||
+ | <html> | ||
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+ | <head> | ||
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+ | <title>Ordered List</title> | ||
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+ | counter-increment: OrderedList; | ||
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+ | ol li :before { | ||
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+ | content: "[" counter(OrderedList) "]"; | ||
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+ | } | ||
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+ | </style> | ||
+ | <html> | ||
+ | <ol> | ||
+ | |||
+ | <li> | ||
+ | <a id="1" href="https://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-5-12" target="_blank"> | ||
+ | BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410</a> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </html> |
Latest revision as of 01:21, 28 October 2020
YmdB regulator of RNAseIII in E. coli with tetA/tetR promoters - RBD - T1 terminator
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 59
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 59
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 59
Illegal BglII site found at 68
Illegal BamHI site found at 673
Illegal XhoI site found at 682 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 59
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 59
- 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