DNA

Part:BBa_K5142045

Designed by: Yuchen Liang   Group: iGEM24_HunanU   (2024-10-01)


Compositive construct for A27L-3Stop mutagenesis

This part was used to replace the wild-type A27L coding sequence in vaccinia virus (VACV) genome with A27L-3Stop, via homologous recombination in VACV genome. This part includes four functional elements in sequence: 1) vaccinia virus A28L, which serves as the left homologous arm; 2) GFP cds regulated by poxviral H5 promoter in reverse direction, which is used to indicate the success of VACV infection and proximity of the construct to viral genome; 3) A27L-3Stop regulated by overlapping early/late poxviral promoter, which is the core sequence for recombination; 4) partial sequence of A26L, which serves as the right homologous arm. The structure of this construct is shown in the following figure.

Figure 1.Schematic diagram showing the structure of the compositive construct for A27L-3Stop mutagenesis. The arrows indicate the directions of transcription and the BioBrick scars are omitted.

This part is composed of the following basic parts aligned tandemly: vaccinia virus A28L (BBa_K5142010), reverse complementary GFP cds (BBa_K5142013), reverse poxviral H5 promoter (BBa_K5142012), overlapping early/late poxviral promoter (BBa_K5142011), vaccinia virus A27L-3stop (BBa_K5142007), vaccinia virus A26L (partial) (BBa_K5142014).

1. To prevent potential readthrough transcription when inserted into vaccinia virus genome, the GFP was transcribed in the direction reverse to the adjacent viral genes (e.g., A26L, A27L and A28L) which was driven by poxviral H5 promoter.
2. Considering the whole A26L sequence is too long as a homologous arm, we chose 500 bp sequence adjacent to A27L only to construct the recombinant element.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 483
    Illegal suffix found in sequence at 462
    Illegal EcoRI site found at 1243
    Illegal EcoRI site found at 1393
    Illegal EcoRI site found at 1510
    Illegal EcoRI site found at 1884
    Illegal XbaI site found at 1258
    Illegal XbaI site found at 1408
    Illegal XbaI site found at 1525
    Illegal XbaI site found at 1899
    Illegal SpeI site found at 1223
    Illegal SpeI site found at 1373
    Illegal SpeI site found at 1490
    Illegal SpeI site found at 1864
    Illegal SpeI site found at 2407
    Illegal PstI site found at 1237
    Illegal PstI site found at 1387
    Illegal PstI site found at 1504
    Illegal PstI site found at 1878
    Illegal PstI site found at 2421
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 483
    Illegal EcoRI site found at 1243
    Illegal EcoRI site found at 1393
    Illegal EcoRI site found at 1510
    Illegal EcoRI site found at 1884
    Illegal SpeI site found at 463
    Illegal SpeI site found at 1223
    Illegal SpeI site found at 1373
    Illegal SpeI site found at 1490
    Illegal SpeI site found at 1864
    Illegal SpeI site found at 2407
    Illegal PstI site found at 477
    Illegal PstI site found at 1237
    Illegal PstI site found at 1387
    Illegal PstI site found at 1504
    Illegal PstI site found at 1878
    Illegal PstI site found at 2421
    Illegal NotI site found at 7
    Illegal NotI site found at 470
    Illegal NotI site found at 489
    Illegal NotI site found at 1230
    Illegal NotI site found at 1249
    Illegal NotI site found at 1380
    Illegal NotI site found at 1399
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 483
    Illegal EcoRI site found at 1243
    Illegal EcoRI site found at 1393
    Illegal EcoRI site found at 1510
    Illegal EcoRI site found at 1884
    Illegal BamHI site found at 697
    Illegal BamHI site found at 1475
    Illegal XhoI site found at 1290
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 463
    Illegal EcoRI site found at 483
    Illegal EcoRI site found at 1243
    Illegal EcoRI site found at 1393
    Illegal EcoRI site found at 1510
    Illegal EcoRI site found at 1884
    Illegal XbaI site found at 498
    Illegal XbaI site found at 1258
    Illegal XbaI site found at 1408
    Illegal XbaI site found at 1525
    Illegal XbaI site found at 1899
    Illegal SpeI site found at 1223
    Illegal SpeI site found at 1373
    Illegal SpeI site found at 1490
    Illegal SpeI site found at 1864
    Illegal SpeI site found at 2407
    Illegal PstI site found at 1237
    Illegal PstI site found at 1387
    Illegal PstI site found at 1504
    Illegal PstI site found at 1878
    Illegal PstI site found at 2421
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal EcoRI site found at 483
    Illegal EcoRI site found at 1243
    Illegal EcoRI site found at 1393
    Illegal EcoRI site found at 1510
    Illegal EcoRI site found at 1884
    Illegal XbaI site found at 16
    Illegal XbaI site found at 498
    Illegal XbaI site found at 1258
    Illegal XbaI site found at 1408
    Illegal XbaI site found at 1525
    Illegal XbaI site found at 1899
    Illegal SpeI site found at 463
    Illegal SpeI site found at 1223
    Illegal SpeI site found at 1373
    Illegal SpeI site found at 1490
    Illegal SpeI site found at 1864
    Illegal SpeI site found at 2407
    Illegal PstI site found at 477
    Illegal PstI site found at 1237
    Illegal PstI site found at 1387
    Illegal PstI site found at 1504
    Illegal PstI site found at 1878
    Illegal PstI site found at 2421
    Illegal NgoMIV site found at 1275
  • 1000
    COMPATIBLE WITH RFC[1000]


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