![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K4574069
AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC
pcDNA3.4 vector containing anti-hTfR single-chain variable fragment attached C-terminally to anti-toxin A (hemolsyin) light and heavy chains of AR-301 (tosatoxumab) monoclonal IgG1 antibody separated by a Gly-Ser-rich linker. This part is commonly referred to as construct H (MUC-AB-H) 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
- APG11 (Part:BBa_K4574020): APG-repeat based linker for fusion of immunoglobulins to single-chain variable fragments (scFv)
- hu128.1-VL (Part:BBa_K4574008): Variable light chain locus targeting human transferrin receptor (hTfR)
- GS18 (Part:BBa_K4574021): GS-rich linker for joining variable fragments in a single-chain variable fragment (scFv)
- hu128.1-VH (Part:BBa_K4574009): Variable heavy chain locus targeting human transferrin receptor (hTfR)
- GS108 (Part:BBa_K4574026): GS-rich linker for joining the antigen-binding fragments with add-ons 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 first insert, comprising Part:BBa_K4574026, the second insert, comprising Part:BBa_K4574008, Part:BBa_K4574021, and Part:BBa_K4574009, and the backbone, comprising Part:BBa_K4574067, PCR fragments were amplified using the following oligonucleotides:
- H1_F_AR301-VH_108aaGS: 5'-ACTTCCGGAACCAGAAGTTCAGATGGTGCAGAGCGG-3'
- E2_R_IgL2-CL_11aaAPG: 5'-GCTCCAGGAAGTGGGACAGGTTCCGGAGCACTCGGTAGGAGCC-3'
- E3_F_hu128.1-VL_11aaAPG: 5'-CCCGGGAGCGGAACCTGTCCCACTTCCGCTATTCAACTGACTCAGAGCCC-3'
- H4_R_hu128.1-VH_108aaGS: 5'-GCTGCTTGTACCTGTGCTACCTGACCCCGACCCAGAGCTCCCGCCTGAGCTCACGGTTACAAGAGTCC-3'
- H5_F_108aaGS_hu128.1-VH: 5'-GTAGCACAGGTACAAGCAGCAG-3'
- H6_R_108aaGS_AR301-VH: 5'-GAAGTTCAACTTCCGGAACCAGATGACCC-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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 9242
Illegal SpeI site found at 8583
Illegal PstI site found at 457
Illegal PstI site found at 1867
Illegal PstI site found at 5214 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 8583
Illegal PstI site found at 457
Illegal PstI site found at 1867
Illegal PstI site found at 5214 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4164
Illegal BglII site found at 8346
Illegal XhoI site found at 1 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 9242
Illegal SpeI site found at 8583
Illegal PstI site found at 457
Illegal PstI site found at 1867
Illegal PstI site found at 5214 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 9242
Illegal SpeI site found at 8583
Illegal PstI site found at 457
Illegal PstI site found at 1867
Illegal PstI site found at 5214
Illegal NgoMIV site found at 553
Illegal NgoMIV site found at 3807
Illegal NgoMIV site found at 4324
Illegal NgoMIV site found at 5665
Illegal NgoMIV site found at 5950
Illegal AgeI site found at 647
Illegal AgeI site found at 3265 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2053
Illegal BsaI.rc site found at 4047
Illegal BsaI.rc site found at 7478
Illegal SapI site found at 6395
Illegal SapI.rc site found at 5514
Illegal SapI.rc site found at 5724
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