Part:BBa_K4574062
AR301-LC
pcDNA3.4 vector containing anti-toxin A (hemolsyin) light chain of AR-301 (tosatoxumab) monoclonal IgG1 antibody. This part is commonly referred to as construct A (MUC-AB-A) 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
- 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_K4574006, and the backbone, comprising Part:BBa_K4574060, PCR fragments were amplified using the following oligonucleotides:
- A1_F_IgL2_CL_AR301-VL: 5'-TGACCGTTCTGGGACAACCTAAGGCCGCTCC-3'
- A2_R_Leader(L)_AR301-VL: 5'-GAGGACGCTTTGGGCCCAAGATCCGGTACAGTG-3'
- A3_F_AR301-VL_Leader(L): 5'-GGCCCACAAAGCGTCCTCACCCAATCCC-3'
- A4_R_AR301-VL_IgL2_CL: 5'-GGACAACCCAGAACGGTCACCTTTGTTCCTGTAC-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 heavy chain Part:BBa_K4574063) 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 6725
Illegal SpeI site found at 6066
Illegal PstI site found at 457
Illegal PstI site found at 2697 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 6066
Illegal PstI site found at 457
Illegal PstI site found at 2697 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1647
Illegal BglII site found at 5829
Illegal XhoI site found at 1 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 6725
Illegal SpeI site found at 6066
Illegal PstI site found at 457
Illegal PstI site found at 2697 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 6725
Illegal SpeI site found at 6066
Illegal PstI site found at 457
Illegal PstI site found at 2697
Illegal NgoMIV site found at 553
Illegal NgoMIV site found at 1290
Illegal NgoMIV site found at 1807
Illegal NgoMIV site found at 3148
Illegal NgoMIV site found at 3433
Illegal AgeI site found at 647
Illegal AgeI site found at 748 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1530
Illegal BsaI.rc site found at 4961
Illegal SapI site found at 3878
Illegal SapI.rc site found at 2997
Illegal SapI.rc site found at 3207
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