Part:BBa_K3362003
Transcriptional Synchronization Cassette (Eukaryotic Version)
On the DNA level, the device is inserted into a naturally occurring gene between its ORF and transcription terminating sequence. The device comprises a synthetic poly-T tail, followed by a 3’ self-cleaving ribozyme, a spacer, a 5’ self-cleaving ribozyme, a variable output and another 3’ self-cleaving ribozyme. When a pre-mRNA is synthesized, everything except the promoter and a part of the terminating sequence is transcribed. During transcription, the poly-T tail is transcribed into a poly-A tail. After transcription, the ribozymes on the pre-mRNA undergo self-cleavage and the ORF gets a 5’ cap. The part containing the ORF is now a functional mRNA and can be exported and translated into a protein due to the 5’ cap and synthetic poly-A tail. The synthetic poly-A tail enables the nuclear export and cytoplasmic translation of the mRNA. If no synthetic poly-A tail would be added, the ribozymes would be likely to prevent natural polyadenylation of the mRNA, which would inhibit nuclear export and cytoplasmic translation. The part that contains the 3’ and 5’ self-cleaving ribozyme as well as the spacer will be degraded by nucleases. The spacer is necessary to give the ribozymes enough space to assume their active secondary structure without interrupting each other. The DNA sequence of the spacer was chosen to be poly T to minimize secondary structure formation and interaction with neighboring sequences. Adenine was chosen for the spacer, so that an interaction with the poly-A tail is as unlikely as possible after transcription: Thymine (on the mRNA) would interact readily with the poly-A tail and act as an antisense inhibitor. Cytosine and guanine might hamper the transcription by being more prone to stay double-stranded. Thus, only Adenine is used for the spacer on the DNA sequence. The output can function independently after being cut out by the ribozymes. The last 3’ self-cleaving ribozyme and the attached part of the transcribed terminating sequence are degraded by nucleases too. The stability of the synchronized part of the 3’ design can be additionally influenced by varying the length of the synthetic poly-T tail: The shorter the tail, the earlier is the synchronized part degraded by the cell.
We plan to use this cassette to synchronize the expression of RFP with a RNAi-mediating molecule (saiRNA or shRNA) to downregulate the expression of genomic YFP in genetically modified Zea mays. This way, we want to proof that transcriptional synchronization of a protein coding sequence with a functional RNA coding sequence can work.
Note: The cassette has to be placed after the ORF of a protein coding sequence and before the terminating sequence. If it should be used to synchronize two functional RNAs, then the poly A tail, first hepatitis delta virus ribozyme and spacer have to be removed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 50
Illegal NgoMIV site found at 79
Illegal NgoMIV site found at 1695
Illegal NgoMIV site found at 1724 - 1000COMPATIBLE WITH RFC[1000]
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