Part:BBa_K4574063
AR301-HC
pcDNA3.4 vector containing anti-toxin A (hemolsyin) heavy chain of AR-301 (tosatoxumab) monoclonal IgG1 antibody. This part is commonly referred to as construct B (MUC-AB-B) 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
- 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_K4574007, and the backbone, comprising Part:BBa_K4574061, PCR fragments were amplified using the following oligonucleotides:
- B1_F_IgG1-CH_AR301-VH: 5'-GGAGCTGACGGTCACCATGCTTCTACCAAGGGACCCAGC-3'
- B2_R_Leader(H)_AR301-VH: 5'-GAAGTTCAGAGCACAAACGCCCTGCAG-3'
- B3_F_AR301-VH_Leader(H): 5'-AGCACAAACGCGAAGTTCAGATGGTGCAGAGCG-3'
- B4_R_AR301-VH_IgG1-CH: 5'-CGGAGCTGACGGTCACCATAGTT-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 co-expressed (together with its respective light chain Part:BBa_K4574062) 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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 7427
Illegal SpeI site found at 6768
Illegal PstI site found at 52
Illegal PstI site found at 3399 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 6768
Illegal PstI site found at 52
Illegal PstI site found at 3399 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2349
Illegal BglII site found at 6531
Illegal XhoI site found at 1 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 7427
Illegal SpeI site found at 6768
Illegal PstI site found at 52
Illegal PstI site found at 3399 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 7427
Illegal SpeI site found at 6768
Illegal PstI site found at 52
Illegal PstI site found at 3399
Illegal NgoMIV site found at 1992
Illegal NgoMIV site found at 2509
Illegal NgoMIV site found at 3850
Illegal NgoMIV site found at 4135
Illegal AgeI site found at 1450 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 238
Illegal BsaI.rc site found at 2232
Illegal BsaI.rc site found at 5663
Illegal SapI site found at 4580
Illegal SapI.rc site found at 3699
Illegal SapI.rc site found at 3909
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