Part:BBa_K4877022

Custom DART VADAR entry sequence with HBB 5' and 3' UTRs

This composite part consists of a custom DART VADAR [1] open reading frame under the control of a T7 promoter. The part was designed from the published DART VADAR sequence as template and includes several changes and modifications. It can be used to generate DART VADAR-based mRNA sensors by in vitro transcription (IVT) with T7 polymerase. An empty sensor cloning site can be used to insert sensor sequences by HindIII digestion followed by Gibson assembly.

Following changes were introduced to the original sequence: Following changes were introduced to the original sequence:

• - A T7 promoter was added.
• - The 5' and 3' UTR were replaced by the UTRs from human beta globin (HBB).
• - The original TagBFP transfection marker was replaced with an mCherry transfection marker.
• - The payload sequence (mNeonGreen) has been flanked with additional SphI and SalI restriction sites, which allows for an easy exchange of the payload using restriction cloning.
• - The PolyA-signal in the original 3'UTR was removed, since this part was designed to be used in IVT applications where the PolyA-tail should be introduced in a subsequent step.
• - A T7 terminator was added.
• - Several illegal restriction sites were silently mutated for RFC10 conformity.

[1] Gayet, R.V., Ilia, K., Razavi, S. et al. Autocatalytic base editing for RNA-responsive translational control. Nat Commun 14, 1339 (2023). https://doi.org/10.1038/s41467-023-36851-z

Characterization – fluorescence microscopy: These results include analysis together with the BBa_K4877021 Part (HBA1).

Fluorescence miscroscopy images of HEK293 cells transfected with mRNA produced by in vitro transcription after 8h, without sensor insert:

Fluorescence miscroscopy images of HEK293 cells transfected with mRNA produced by in vitro transcription after 24h, without sensor insert:

Fluorescence miscroscopy images of HEK293 cells transfected with mRNA produced by in vitro transcription after 48h, without sensor insert:

Characterization – FACS: These results include analysis together with the BBa_K4877021 Part (HBA1).

The red signal represents the untreatened negativ control of HEK cell. The yellow signal represents the pASTERISK_HBA generated mRNA molecules. The blue signal represents the HBA variant in different Lipofectamin concentration. The light green seignal represents die HBB Variant with lower Lipofectamin concentration in comparison to the dark green singal, which represent the HBB Variant transfection with lipofectamin. All detections were performed 72 h after transfection.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 890
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 839
  Illegal BamHI site found at 1143
  Illegal BamHI site found at 3320
  Illegal XhoI site found at 882
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2154