Regulatory

Part:BBa_K1484336:Design

Designed by: Angelina Munabi   Group: iGEM14_Cambridge-JIC   (2014-10-10)

P_NiI20, marchantia promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 670
    Illegal EcoRI site found at 832
    Illegal XbaI site found at 49
    Illegal XbaI site found at 1142
    Illegal PstI site found at 79
    Illegal PstI site found at 722
    Illegal PstI site found at 1271
    Illegal PstI site found at 1721
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 670
    Illegal EcoRI site found at 832
    Illegal PstI site found at 79
    Illegal PstI site found at 722
    Illegal PstI site found at 1271
    Illegal PstI site found at 1721
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 670
    Illegal EcoRI site found at 832
    Illegal BglII site found at 1961
    Illegal BamHI site found at 880
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 670
    Illegal EcoRI site found at 832
    Illegal XbaI site found at 49
    Illegal XbaI site found at 1142
    Illegal PstI site found at 79
    Illegal PstI site found at 722
    Illegal PstI site found at 1271
    Illegal PstI site found at 1721
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 670
    Illegal EcoRI site found at 832
    Illegal XbaI site found at 49
    Illegal XbaI site found at 1142
    Illegal PstI site found at 79
    Illegal PstI site found at 722
    Illegal PstI site found at 1271
    Illegal PstI site found at 1721
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1623
    Illegal BsaI.rc site found at 813
    Illegal SapI site found at 161


Design Notes

The removal of illegal restriction sites was considered but not completed due to direct interaction of the DNA with regulatory proteins. Also, it was ensured that the region selected for this part was directly upstream of the first ATG of the gene used to identify the promoter sequence. This was so that the 5' UTR, that is vital in plants, was maintained.


Source

This part was found by screening the genome of the Marchantia polymorpha Cam strain maintained by the Haseloff lab for homologues to Nitrate Transporter proteins in the following organisms and including corresponding genes: A. thaliana - AtNRT2.1, AtNRT2.2; N. plumbaginifolia - NpNRT2; C. reinhardtii - CrNRT2.1, CrNRT2.3; P. patens - NRT2.1, NRT2.2, NRT2.3, NRT2.4, NRT2.5

Transcription is thought to be induced by nitrates. Matches to the protein CDS sequence were found using Geneious to perform a tblastn search on the genome scaffolds. Predicted genes that contained hits graded above 30% and with at least 40% congruence to mRNA transcript sequences were shortlisted. The best gene candidates (judged according to number and distribution of hits along its length, and supporting mRNA sequence) formed the basis for our predicted promoters. This part was isolated from a 2kb region upstream of the first ATG of such a gene.


References

Forde BG. 2000. Nitrate transporters in plants: structure, function and regulation. (BBA) – Biomembranes 1465(1-2):219-235. Filleur S, Daniel-Vedele F. 1998. Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display. Planta 207: 461-469. ThaleMine, id AT1G08090 GenBank, Acc. No. AF019749.1 GenBank, Acc No. Y08210 GenBank, Acc No. AF047718 GenBank, Acc No. Z25438 GenBank, Acc No. AJ223296 http://www.ncbi.nlm.nih.gov/gene/5931334 http://www.ncbi.nlm.nih.gov/gene/5931336 http://www.uniprot.org/uniprot/Q76C04 http://www.uniprot.org/uniprot/Q76C03 http://www.uniprot.org/uniprot/Q76C02