Composite

Part:BBa_K4825059

Designed by: Ye Siyi   Group: iGEM23_GreatBay-SCIE   (2023-10-12)


pTEF2-CDC9-LEU1-tSSA1-pPGK1-COX6-LEU2-tPGK1-pTDH3-COX4-LEU4^S547∆-tTDH1

CDC9 gene from yeast encodes the activity required during DNA replication by DNA ligase I. The N- terminal localization signals from CDC9 was used to taget Leu1p in the mitochondria, the first 47 amino acids from CDC9 were appended to the N- terminus of the open reading frame. LEU1 is an endogenous gene in yeast 2-ketoacid elongation pathway. It encodes for isopropylmalate Isomerase, which isomerizes 2-isopropylmalate(2-IPM), an intermediate in the pathway, to produce 3-isopropylmalate(3-IPM). isopropylmalate Isomerase naturally functions in cytosol.

LEU2, encoding IPM dehydrogenase, is an endogenous gene in yeast 2-ketoacid elongation pathway. The enzyme oxidizes and decarboxylates 3 IPM to produce 2-ketoisocaproate(KIC), and important precursor for isoamyl acetate. IPM dehydrogenase naturally function in cytosol.

LEU4 encodes for 2-isopropylmalate synthase(2-IPMS), which acetylates KIV and turning it into 2-isopropylmalate(2-IPM). This is one of the three reaction in 2-ketoacid elongation pathway. 2-IPMS naturally locates both in mitochondria and cytosol.

The terminal enzyme in the mitochondrial respiratory chain is COX. COX contains 13 mitochondrial and nuclear encoded subunits. The nuclear encoded subunits have yet unknown function but are hypothesized to play a role in constructing and regulating the complex. The mitochondrial encoded subunits aids in the performance of the ETC and proton pumping activities. The N- terminal localization signals from COXIV is used to target Leu4p to mitochondria, and the first 26 amino acids from COX4 appends to the N-terminus of the corresponding open reading frame.

COX6 is the last enzyme in line in the mitochondrial electron transport chain, driving oxidative phosphorylation. it is a component of the cytochrome c oxidase. The N- terminal localization signals from COXVI were used to target Leu2p to mitochondria, and the first 41 amino acids from the COX VI were appended to the N- terminus of the corresponding open reading frame.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal NheI site found at 9453
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal BglII site found at 6405
    Illegal BglII site found at 10660
    Illegal BamHI site found at 9606
    Illegal BamHI site found at 10572
    Illegal XhoI site found at 5048
    Illegal XhoI site found at 11251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
    Illegal NgoMIV site found at 2843
    Illegal AgeI site found at 425
    Illegal AgeI site found at 7131
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5034
    Illegal BsaI site found at 5303
    Illegal BsaI.rc site found at 5284
    Illegal BsaI.rc site found at 6415
    Illegal SapI site found at 4305
    Illegal SapI site found at 6865
    Illegal SapI.rc site found at 6849


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