Plasmid

Part:BBa_K4231018:Design

Designed by: Shi Han   Group: iGEM22_USTC   (2022-09-30)


pUC19 carrying with IDI, NADH-HMG, ERG8, ERG19 and URA3 tag from Y.lipolytica


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2325
    Illegal EcoRI site found at 8712
    Illegal PstI site found at 862
    Illegal PstI site found at 1992
    Illegal PstI site found at 7315
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2325
    Illegal EcoRI site found at 8712
    Illegal NheI site found at 634
    Illegal PstI site found at 862
    Illegal PstI site found at 1992
    Illegal PstI site found at 7315
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2325
    Illegal EcoRI site found at 8712
    Illegal BglII site found at 719
    Illegal BglII site found at 6238
    Illegal BamHI site found at 974
    Illegal BamHI site found at 1073
    Illegal BamHI site found at 1613
    Illegal BamHI site found at 6936
    Illegal BamHI site found at 8102
    Illegal XhoI site found at 3028
    Illegal XhoI site found at 3862
    Illegal XhoI site found at 3895
    Illegal XhoI site found at 6653
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2325
    Illegal EcoRI site found at 8712
    Illegal PstI site found at 862
    Illegal PstI site found at 1992
    Illegal PstI site found at 7315
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2325
    Illegal EcoRI site found at 8712
    Illegal PstI site found at 862
    Illegal PstI site found at 1992
    Illegal PstI site found at 7315
    Illegal NgoMIV site found at 638
    Illegal NgoMIV site found at 1366
    Illegal NgoMIV site found at 2247
    Illegal NgoMIV site found at 2463
    Illegal NgoMIV site found at 2967
    Illegal NgoMIV site found at 3219
    Illegal NgoMIV site found at 3531
    Illegal AgeI site found at 2343
    Illegal AgeI site found at 3540
    Illegal AgeI site found at 3627
    Illegal AgeI site found at 4153
    Illegal AgeI site found at 8270
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 7991
    Illegal BsaI.rc site found at 2386
    Illegal BsaI.rc site found at 3048
    Illegal BsaI.rc site found at 3396
    Illegal BsaI.rc site found at 7710
    Illegal BsaI.rc site found at 8060


Design Notes

At the C-terminus of the IDI, NADH-HMG, ERG8 and ERG19 enzyme genes, we added the PTS1 sequence ( GGGSSKL ) to ensure that the enzyme protein can be localized in the peroxisome. When assembling the plasmid, we chose In-Fusion® Snap Assembly Master Mix to successfully complete the construction.


Source

The coding sequences of IDI, ERG8, ERG19, URA3 were amplified from Y.lipolytica.The ORF of NADH-HMG was found in NCBI and was synthesized according to the sequence.

References