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Revision as of 09:21, 4 October 2023


CYP81A10V7 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP81A10V7 (BBa_K4806005), mNeonGreen (BBa_K4806006) and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).

The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
    Illegal PstI site found at 3519
    Illegal PstI site found at 3926
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
    Illegal PstI site found at 3519
    Illegal PstI site found at 3926
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3535
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
    Illegal PstI site found at 3519
    Illegal PstI site found at 3926
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
    Illegal PstI site found at 3519
    Illegal PstI site found at 3926
    Illegal NgoMIV site found at 2274
    Illegal NgoMIV site found at 4719
    Illegal AgeI site found at 268
    Illegal AgeI site found at 3864
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We confirmed that this construct is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYP81A10V7 level 2 with mNeonGreen
We digested this level 2 MoClo part with the restriction enzymes EcoRV, NdeI and XhoI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.


Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.