Part:BBa_K4574067

AR301-LC-57aaGS-HC

pcDNA3.4 vector containing anti-toxin A (hemolsyin) heavy and light chains of AR-301 (tosatoxumab) monoclonal IgG1 antibody separated by a Gly-Ser-rich linker. This part is commonly referred to as construct F (MUC-AB-F) in the context of Team Munich's iGEM 2023 project.

Design

This construct is comprised of the following components:

• XhoI (Part:BBa_K4574029): XhoI restriction site, allows for insert extraction
• Kozak (Part:BBa_K4574027): Consensus sequence which enhances the initiation of translation
• hVL-Leader (Part:BBa_K4574001): Human light chain lambda leader sequence for antibody secretion
• AR-301-VL (Part:BBa_K4574006): Variable light chain locus targeting toxin A (hemolysin) of Staphylococcus aureus
• hIG-CL (Part:BBa_K4574018): Human immunoglobulin light chain constant lambda region
• GS57 (Part:BBa_K4574022): GS-rich linker for joining the antigen-binding fragments in a single-chain fragment (scFab)
• hVH-Leader (Part:BBa_K4574002): Human heavy chain leader sequence for antibody secretion
• AR-301-VH (Part:BBa_K4574007): Variable heavy chain locus targeting toxin A (hemolysin) of Staphylococcus aureus
• hIG-CH (Part:BBa_K4574019): Human immunoglobulin heavy chain constant gamma 1 region
• 2xStop (Part:BBa_K4574028): Double stop codon sequence ensuring enhanced translation termination
• SacI (Part:BBa_K4574030): SacI restriction site, allows for insert extraction
• pcDNA3.4 (Part:BBa_K4574000): pcDNA3.4-TOPO backbone for constitutive high-level transgene expression in mammalian systems

Cloning

The cloning of this part has been performed using Gibson (scarless) assembly in DH5α E. coli competent cells. The insert, comprising Part:BBa_K4574022, and the backbone, comprising Part:BBa_K4574064, PCR fragments were amplified using the following oligonucleotides:

• F1_F_AR301-VH_57aaGS: 5'-CGAGGCCCCAGCAGAAGTTCAGATGGTGCAGAGCGG-3'
• F2_R_IgL2-CL_57aaGS: 5'-GGCGGGAGCTCTGGGTCGGGGTCAGGTAGCACAGGTACAAGGGAGCACTCGGTAGGAGCCA-3'
• F3_F_57aaGS_IgL2-CL: 5'-GTCAGGTAGCACAGGTACAAGCA-3'
• F4_R_57aaGS_AR301-VH: 5'-GAAGTTCAGATGCGAGGCCCCAGCAGTTGC-3'

The provided oligonucleotides include a binding region (indicated by an underline) as well as an overhang required by the assembly method.

Usage and Biology

This construct has been used in mammalian cell culture (HEK293T, Exi293, RAMOS). Specifically, it has been expressed using a high-yield Expi293 expression system (Gibco, a brand of Thermo Fisher Scientific, Braunschweig, Germany; catalog number: A14635) and purified in FPLC using Protein A agarose cartridges (ibidi, Goettingen, Germany; catalog number: 6-2022-001).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 8306
  Illegal SpeI site found at 7647
  Illegal PstI site found at 457
  Illegal PstI site found at 931
  Illegal PstI site found at 4278
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 7647
  Illegal PstI site found at 457
  Illegal PstI site found at 931
  Illegal PstI site found at 4278
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3228
  Illegal BglII site found at 7410
  Illegal XhoI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 8306
  Illegal SpeI site found at 7647
  Illegal PstI site found at 457
  Illegal PstI site found at 931
  Illegal PstI site found at 4278
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 8306
  Illegal SpeI site found at 7647
  Illegal PstI site found at 457
  Illegal PstI site found at 931
  Illegal PstI site found at 4278
  Illegal NgoMIV site found at 553
  Illegal NgoMIV site found at 2871
  Illegal NgoMIV site found at 3388
  Illegal NgoMIV site found at 4729
  Illegal NgoMIV site found at 5014
  Illegal AgeI site found at 647
  Illegal AgeI site found at 2329
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1117
  Illegal BsaI.rc site found at 3111
  Illegal BsaI.rc site found at 6542
  Illegal SapI site found at 5459
  Illegal SapI.rc site found at 4578
  Illegal SapI.rc site found at 4788