Composite

Part:BBa_M50047:Design

Designed by: Michelle Bae   Group: Stanford BIOE44 - S11   (2016-12-08)
Revision as of 06:43, 12 December 2016 by Mbuckup (Talk | contribs) (→‎Design Notes)


Linear DNA to be used with HIV I Integrase pD649


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1873
    Illegal SpeI site found at 200
    Illegal PstI site found at 874
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
    Illegal SpeI site found at 200
    Illegal PstI site found at 874
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1853
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1873
    Illegal SpeI site found at 200
    Illegal PstI site found at 874
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1873
    Illegal SpeI site found at 200
    Illegal PstI site found at 874
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 936
    Illegal SapI.rc site found at 1134


Design Notes

When the HIV-1 genome is integrated into a genome, the integrase protein recognizes specific sequences at the ends of the 5’ and 3’ long terminal repeats, or U3 and U5 region, respectively. U5 sequence recognized by the integrase is 5’-CCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3’, and U3 sequence, also recognized by the integrase is 5’-ACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATC-3’. Accordingly, the ends of our linear DNA contain the terminal recognition regions from either the U5 or U3 region. While the interactions between integrase and the specific bases of these regions are well studied, and it has been shown that only approximately 20 base pairs are necessary for the protein to bind, we decided to use the terminal 40 base pairs because previous similar experiments have shown that this may increase the efficiency of integration. In between the integrase recognition regions, we have all the necessary elements for the expression of another reporter protein, in this case a red fluorescent protein, or RFP. These elements will be sourced from another plasmid so their exact nature is not yet known, but most certainly these elements will include a strong constitutive promoter, a Kozak sequence, the code for RFP, and a terminator.

Glycerol freezer stock information for the Integrase plasmid this composite part interacts with:

Plasmid name: pD649_Integrase

DNA 2.0 Gene #: 273871

Organism: HEK 293T

Device type: actuator

Barcode #'s: 0133027143, 0133027110, 0133027160

Box label: BIOE44 F16

Source

U5 and U3 sequences were sourced from the links in the reference section. mRuby3 was sourced from the Parts Registry (BBa_M50009).

References

Mizuarai S, et al. 1999. Integrase-mediated nonviral gene transfection with enhanced integration efficiency. J Biosci Bioeng. 88(5):461-7.

Kessl, J.J., et al. 2009. HIV-1 Integrase-DNA Recognition Mechanisms. Viruses. 1(3):713–736.

Esposito D., Craigie R. 1998. Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction. EMBO J. 17(19):5832-43.

2006. HIV-1, complete genome. GenBank:AF033819.3.