Ath-HEN1::T2A::mCherry::TY1::H2B-pCFJ350-ttTi5605
(Plasmid #108362)

• Purpose: Expression of 2xFlag MYC tagged A.thaliana-HEN1 (Ath-HEN1, codon optimised for C. elegans) and mCherry under the promoter of choice in C. elegans
• Depositing Lab: Luisa Cochella
• Publication: Alberti et al Nat Methods. 2018 Feb 26. pii: nmeth.4610. doi: 10.1038/nmeth.4610.

Backbone

• Vector backbone: pCFJ350-ttTi5605
• Backbone size w/o insert (bp): 7517
• Total vector size (bp): 12670
• Modifications to backbone: The cassette for bicistronic expression of Ath-HEN1 and mCherry-H2B was inserted into pCFJ350-ttTi5605 by Gibson cloning between the M13 FWD and M13 REV priming sites.
• Vector type: Worm Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Ath-HEN1
• Alt name: A. thaliana HEN1
• Species: A. thaliana (mustard weed); Codon optimised for C. elegans
• Insert Size (bp): 2979
• Entrez Gene: HEN1 (a.k.a. AT4G20910, CORYMBOSA 2, CRM2, HUA ENHANCER 1, T13K14.70, T13K14_70)
• Promoter: Ath-HEN1 coding sequence
• Tags / Fusion Proteins:
  • 2xFlag (N terminal on insert)
  • MYC (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CCGGCCggcgcgccGCTAGCcaccatggattacaaggatgacg
• 3′ sequencing primer: GAGGTCGGTCTTCTTCTTCTCAAC

Gene/Insert 2

• Gene/Insert name: mCherry
• Insert Size (bp): 708
• Promoter: mCherry
• Tag / Fusion Protein:
  • T2A peptide (N terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: AGAAGAAGAAGACCGACCTCgagggcagaggaagtctgc
• 3′ sequencing primer: TCCTGATTGGTATGCACTTCcttgtacagctcgtccatgcc

Gene/Insert 3

• Gene/Insert name: his-15
• Alt name: histone sequence
• Species: C. elegans (nematode)
• Insert Size (bp): 369
• Entrez Gene: his-15 (a.k.a. CELE_ZK131.9)
• Promoter: H2B
• Tag / Fusion Protein:
  • TY1 linker (N terminal on insert)

Cloning Information for Gene/Insert 3

• Cloning method: Gibson Cloning
• 5′ sequencing primer: GAAGTGCATACCAATCAGGACCC
• 3′ sequencing primer: CTCAGATATCAATACCATGCCTATTACTTGCTGGAAGTGTACTTGG

Gene/Insert 4

• Gene/Insert name: unc-54 3' UTR
• Species: C. elegans (nematode)
• Insert Size (bp): 733
• Promoter: A rab-3:Ath-HEN1:unc-54 3' UTR was cloned as a unique AvrII fragment from an intermediate vector into pCFJ350-ttTi5605

Cloning Information for Gene/Insert 4

• Cloning method: Restriction Enzyme
• 5′ cloning site: AvrII (not destroyed)
• 3′ cloning site: AvrII (not destroyed)
• 5′ sequencing primer: TTTTCCTAGGGATTACGCCAAGCTTGCATGC
• 3′ sequencing primer: AAAACCTAGGCACCGTCATCACCGAAACGC

Resource Information

• Supplemental Documents:
  • Annotated GenBank file

Terms and Licenses

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