Part:BBa_K4574064

AR301-LC-2A-HC

pcDNA3.4 vector containing anti-toxin A (hemolsyin) heavy and light chains of AR-301 (tosatoxumab) monoclonal IgG1 antibody separated by a T2A ribosome skipping site. This part is commonly referred to as construct C (MUC-AB-C) 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
• T2A (Part:BBa_K4574026): 2A-like ribosomal skipping sequence for equimolar co-expression of two separate polypeptides from a single mRNA
• 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_K4574063, and the backbone, comprising Part:BBa_K4574062, PCR fragments were amplified using the following oligonucleotides:

• C1_F_pcDNA_IgG1-CH: 5'-TCATCACTTGGAGCTCAAGGGTTCGATCCCTACC-3'
• C2_R_IgL2-CL_2A: 5'-GGATCTGGCGAAGGCAGAGGATCTCTGGGAGCACTCGGTAGGAGCC-3'
• C3_F_Leader(H)_2A: 5'-AGGTCCAGGGTTCTCTTCCACGTCGCCGCATGTCAGCAGAGATCCTCTGCCTTCGATGGGCTCTACAGCTATTCTGGG-3'
• C4_R_IgG1-CH_pcDNA: 5'-GAGCTCAAGGGTCATCACTTGCCAGGGGACAGA-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 8198
  Illegal SpeI site found at 7539
  Illegal PstI site found at 457
  Illegal PstI site found at 823
  Illegal PstI site found at 4170
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 7539
  Illegal PstI site found at 457
  Illegal PstI site found at 823
  Illegal PstI site found at 4170
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3120
  Illegal BglII site found at 7302
  Illegal XhoI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 8198
  Illegal SpeI site found at 7539
  Illegal PstI site found at 457
  Illegal PstI site found at 823
  Illegal PstI site found at 4170
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 8198
  Illegal SpeI site found at 7539
  Illegal PstI site found at 457
  Illegal PstI site found at 823
  Illegal PstI site found at 4170
  Illegal NgoMIV site found at 553
  Illegal NgoMIV site found at 2763
  Illegal NgoMIV site found at 3280
  Illegal NgoMIV site found at 4621
  Illegal NgoMIV site found at 4906
  Illegal AgeI site found at 647
  Illegal AgeI site found at 2221
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1009
  Illegal BsaI.rc site found at 3003
  Illegal BsaI.rc site found at 6434
  Illegal SapI site found at 5351
  Illegal SapI.rc site found at 4470
  Illegal SapI.rc site found at 4680