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Part:BBa_K4800003
Ptrc-sfp-MmCAR-YahK
(1)Ptrc is a promoter.(2)YahK is a acyl-CoA thioester hydrolase from Haemophilus influenzae.(3) Sfp is a phosphopantetheinyl transferase from Bacilus subtilis.(4) MmCAR is a Carboxylic acid reductase from Mycobacterium marinum.(5) YahK is a NADPH-dependent aldehyde reductase.
Build:
1.Experimental Methods
1.1 Construction of PCWJ-YciA-sfp-MmCAR-YahK
The pCWJ plasmid was extracted from a laboratory conserved strain. In-Fusion cloning was used to ligate the above vector to the Ptrc-YciA-sfp-MmCAR-YahK fragment.
1.2 Construction of PRSFDuet-YciA-sfp-MmCAR-YahK
The PRSFDuet plasmid was extracted from laboratory conserved strains. The fragment Ptrc-YciA-sfp-MmCAR-sfp-Yahk was amplified by PCR using PCWJ-YciA-sfp-MmCAR-YahK as a template, and Ptrc-YciA-MmCAR-sfp-Yahk was ligated by In-fusion cloning to the vector PRSFDuet that was linearized using PCR for homologous recombination ligation.
Result
By comparing the fermentation production of 1,5-PDO content with the previous KA30 with PCWJ+PTRC99a, we can clearly see that KA30 with PRSFDuet+PTRC99a produced more 1,5-PDO, therefore, we decided to use both vectors, PRSFDuet and PTRC99a, in all our future experiments.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1332
Illegal PstI site found at 1767
Illegal PstI site found at 1794
Illegal PstI site found at 3063
Illegal PstI site found at 3090
Illegal PstI site found at 3483
Illegal PstI site found at 3979
Illegal PstI site found at 4623 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1332
Illegal NheI site found at 4891
Illegal PstI site found at 1767
Illegal PstI site found at 1794
Illegal PstI site found at 3063
Illegal PstI site found at 3090
Illegal PstI site found at 3483
Illegal PstI site found at 3979
Illegal PstI site found at 4623 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1332
Illegal BglII site found at 2321
Illegal BglII site found at 2947
Illegal BglII site found at 3223
Illegal BglII site found at 3914
Illegal BamHI site found at 1304
Illegal XhoI site found at 4863 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1332
Illegal PstI site found at 1767
Illegal PstI site found at 1794
Illegal PstI site found at 3063
Illegal PstI site found at 3090
Illegal PstI site found at 3483
Illegal PstI site found at 3979
Illegal PstI site found at 4623 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1332
Illegal PstI site found at 1767
Illegal PstI site found at 1794
Illegal PstI site found at 3063
Illegal PstI site found at 3090
Illegal PstI site found at 3483
Illegal PstI site found at 3979
Illegal PstI site found at 4623
Illegal NgoMIV site found at 3591
Illegal NgoMIV site found at 3653
Illegal NgoMIV site found at 3870
Illegal NgoMIV site found at 4317
Illegal AgeI site found at 1497
Illegal AgeI site found at 2508
Illegal AgeI site found at 2805
Illegal AgeI site found at 3690
Illegal AgeI site found at 3699
Illegal AgeI site found at 4374 - 1000COMPATIBLE WITH RFC[1000]