LA-ELP-ybbR-RLC-ddfln4-Xmod-DocIII-Ctag
(Plasmid #257335)

• Purpose: Polyprotein for applying force to the N- and C-termini of the RLC in the RLC complex of human β-cardiac myosin
• Depositing Lab: Tom Perkins
• Publication: Deopa et al ACS Nano. 2026 Jun 9;20(22):15982-16000. doi: 10.1021/acsnano.5c22720. Epub 2026 May 21.

Backbone

• Vector backbone: pET28
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5236
• Total vector size (bp): 7969
• Vector type: Mammalian Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: RLC
• Alt name: Human Myosin Regulatory Light chain 2
• Species: H. sapiens (human)
• Insert Size (bp): 498
• GenBank ID: NP_000423.2
• Entrez Gene: MYL2 (a.k.a. CMH10, MFM12, MLC-2, MLC-2s/v, MLC-2v, MLC2)
• Promoter: T7
• Tags / Fusion Proteins:
  • ELP120 (Elastin-Like Polypeptides)
  • TEV (TEV cleavage site)
  • ybbR
  • ddfln4
  • Xmod

Cloning Information for Gene/Insert 1

• Cloning method: Other
• 5′ sequencing primer: Whole plasmid nanopore sequencing
• 3′ sequencing primer: Whole plasmid nanopore sequencing

Gene/Insert 2

• Gene/Insert name: Lever Arm
• Alt name: β-cardiac myosin heavy chain 7
• Species: H. sapiens (human)
• Insert Size (bp): 111
• Mutation: only amino acids # 810-846 are used
• GenBank ID: NP_000248.2
• Entrez Gene: MYH7 (a.k.a. CMD1S, CMH1, CMYO7A, CMYO7B, CMYP7A, CMYP7B, MPD1, MYHCB, SPMD, SPMM)
• Promoter: T7
• Tags / Fusion Proteins:
  • Dockerin III
  • Ctag (C terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Other
• 5′ sequencing primer: Whole plasmid nanopore sequencing
• 3′ sequencing primer: Whole plasmid nanopore sequencing

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Lever arm = [β-cardiac myosin heavy chain 7 (NP_000248.2, residues 810–846)].

ELP120 = Elastin-Like Polypeptides. A 120-nm unstructured polypeptide (60x VPGXG) linker.

TEV = TEV protease cleavage site

ybbR = An 11-amino acid peptide tag that is enzymatically conjugated to a Coenzyme A-derivatized surface via Sfp phosphopantetheinyl transferase.

RLC = Human cardiac myosin regulatory light chain 2 (NP_000423.2).

ddFLN4 = The fourth immunoglobulin (Ig) domain of the F-actin cross-linking protein filamin from the slime mold Dictyostelium discoideum. It unfolds with a characteristic two-step mechanical fingerprint that is used to identify single-molecule attachments in AFM assays.

Xmod = A protein domain that serves as a mechanical stabilizer to strengthen the bond between the adjacent dockerin to cohesin.

Dockerin III = A calcium-binding domain that acts as the "plug" to the cohesin (Coh) "socket". The Xmod-DocIII:cohesin complex is one of the strongest non-covalent interactions known and is typically used in single-molecule, force-spectroscopy assays to attach surface anchor polyproteins protein to atomic-force-microscopy cantilevers.

Ctag = an 8-amino acid C-terminal protein tag used for protein purification. It binds to PDZ-sepharose and can be competed off using a 5 amino acid WETWV peptide.