vYM133_PB-pEF-osTIR1-T2A-iaaH-T2A-mGFPmut3-mAID-iCasp9-T2A-mCh-AID-BlastR
(Plasmid #160046)

• Purpose: For expressing the full paradoxical circuit
• Depositing Lab: Michael Elowitz
• Publication: Ma et al Cell. 2022 Mar 17;185(6):967-979.e12. doi: 10.1016/j.cell.2022.01.026. Epub 2022 Mar 1.

Backbone

• Vector backbone: PiggyBac
• Backbone manufacturer: SYSTEM BIOSCIENCES
• Vector type: Mammalian Expression, Synthetic Biology
• Selectable markers: Hygromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: osTIR1
• Species: Oryza Sativa (Asian rice)
• Insert Size (bp): 1725
• Promoter: pEF1s

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ATGACATACTTTCCTGAAGAGGTCGTC
• 3′ sequencing primer: CAGAATCTTCACAAAGTTGGGAGC

Gene/Insert 2

• Gene/Insert name: iaaH
• Insert Size (bp): 1401
• Promoter: no promoter, T2A is used

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: cttggtaccgagctcggatccaactcgaggccaccatgGTAGCCATCACGTCACTGGCCC
• 3′ sequencing primer: CCACATCCCCGCAAGTAAGCAGTGATCCGCGTCCCTCggtGTTCGGAAGTCCAGCGAAAT

Gene/Insert 3

• Gene/Insert name: iCasp9
• Species: H. sapiens (human)
• Insert Size (bp): 1236
• Promoter: no promoter, T2A is used
• Tags / Fusion Proteins:
  • mGFPmut3 (N terminal on insert)
  • mAID (N terminal on insert)

Cloning Information for Gene/Insert 3

• Cloning method: Gibson Cloning
• 5′ sequencing primer: atggctagcaaaggtGAGGAG
• 3′ sequencing primer: gtcgagtgcgtagtctggtacgtcgtacggatag

Gene/Insert 4

• Gene/Insert name: BlastR
• Alt name: BSD
• Species: Synthetic; Aspergillus terreus
• Insert Size (bp): 396
• Promoter: no promoter, T2A is used
• Tags / Fusion Proteins:
  • mCherry (N terminal on insert)
  • mAID (N terminal on insert)

Cloning Information for Gene/Insert 4

• Cloning method: Gibson Cloning
• 5′ sequencing primer: gtgagcaagggcgaggagg
• 3′ sequencing primer: taagctgcaataaacaagttgTCAgccctcccacacataacca

Resource Information

• Supplemental Documents:
  • vym133_pb-pef-ostir1-t2a-iaah-t2a-mgfpmut3-maid-icasp9-t2a-mch-aid-blastr.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • piggyBac Limited Use Label License
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

osTIR1 is cloned from #72834
mAID is cloned from #72827
iCasp9 is cloned from #15567