Coding

Part:BBa_K4806004

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-18)
Revision as of 11:45, 20 September 2023 by Lulang (Talk | contribs)


CYP9Q3 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP9Q3 (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing CYP9Q3 using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP9Q3 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806222)
  • 2. CYP9Q3 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806223)
  • 3. CYP9Q3 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806224)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP9Q3 coding sequence the constructs contain the AβSAP(i)-promotor (BBa_K4806013), either the HA-tag (BBa_K3002017)* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)*. 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 1249
    Illegal PstI site found at 1429
    Illegal PstI site found at 1935
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1249
    Illegal PstI site found at 1429
    Illegal PstI site found at 1935
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1791
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1249
    Illegal PstI site found at 1429
    Illegal PstI site found at 1935
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1249
    Illegal PstI site found at 1429
    Illegal PstI site found at 1935
    Illegal NgoMIV site found at 2206
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

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Categories
Parameters
None