Part:BBa_K4879008
CvFAP Expression Construct
The transcriptional unit coding for CvFAP, designed for expression in Y. lipolytica.
The expression cassette was assembled together along with the JcFATB and the JcFATA expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.
Characterization
Cloning: The assembled gene(s) were then inserted into the pYLEX plasmid (sourced from the Yarrowia Lipolytica Expression Kit, procured from Yeastern Biotech), by the means of NEBuilder HiFi DNA Assembly. The expected clones were then digested with NotI and SalI. The resulting digestion product is run on a 0.8% agarose gel. The cloning was confirmed, with the cloning success also being verified with sequencing. The linearized fragment was expected to be at above the 10 kbp band in the ladder (~13,000 bp)
Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.
Expression: To verify the expression of JcFATA, the coding sequence had a 6xHis tag attached to the C-terminus. Subsequently, a Western blot was done for the protein after normalization.
The desired bands were visible in the blot but in the control too, as a result of possible contamination in the control. Thus, further experiments are needed to troubleshoot the problem and characterize the part fully. Depending on the refined results of the experiments, the functional validity of the part will be confirmed for future use.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 2320
Illegal suffix found in sequence at 2590
Illegal EcoRI site found at 2584
Illegal PstI site found at 790
Illegal PstI site found at 1222
Illegal PstI site found at 1489 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2320
Illegal EcoRI site found at 2584
Illegal SpeI site found at 2591
Illegal PstI site found at 790
Illegal PstI site found at 1222
Illegal PstI site found at 1489
Illegal PstI site found at 2605
Illegal NotI site found at 2326
Illegal NotI site found at 2598 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2320
Illegal EcoRI site found at 2584
Illegal XhoI site found at 1955 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 2320
Illegal suffix found in sequence at 2591
Illegal EcoRI site found at 2584
Illegal PstI site found at 790
Illegal PstI site found at 1222
Illegal PstI site found at 1489 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 2320
Illegal EcoRI site found at 2584
Illegal XbaI site found at 2335
Illegal SpeI site found at 2591
Illegal PstI site found at 790
Illegal PstI site found at 1222
Illegal PstI site found at 1489
Illegal PstI site found at 2605
Illegal NgoMIV site found at 2110
Illegal AgeI site found at 2086 - 1000COMPATIBLE WITH RFC[1000]
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