![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K4574068
scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC
pcDNA3.4 vector containing anti-hTfR single-chain variable fragment attached N-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 G (MUC-AB-G) 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
- 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)
- APG11 (Part:BBa_K4574020): APG-repeat based linker for fusion of immunoglobulins to single-chain variable fragments (scFv)
- 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_K4574008, Part:BBa_K4574021, and Part:BBa_K4574009, and the backbone, comprising Part:BBa_K4574067, PCR fragments were amplified using the following oligonucleotides:
- D1_F_AR301-VL_11aaAPG: 5'-CCCGGGAGCGGAACCTGTCCCACTTCCAAAGCGTCCTCACCCAATCC-3'
- D2_R_Leader(L)_hu128.1-VL: 5'-GGCCCAAGATCCGGTACAGTG-3'
- D3_F_hu128.1-VL_Leader(L): 5'-GGCCCAAGATCCGGTACAGCTATTCAACTGACTCAGAGCCCTTC-3'
- D4_R_hu128.1-VH_11aaAPG: 5'-GCTCCAGGAAGTGGGACAGGTTCCGCTTGAGCTCACGGTTACAAGAGTCC-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 9089
Illegal SpeI site found at 8430
Illegal PstI site found at 1240
Illegal PstI site found at 1714
Illegal PstI site found at 5061 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 8430
Illegal PstI site found at 1240
Illegal PstI site found at 1714
Illegal PstI site found at 5061 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4011
Illegal BglII site found at 8193
Illegal XhoI site found at 1 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 9089
Illegal SpeI site found at 8430
Illegal PstI site found at 1240
Illegal PstI site found at 1714
Illegal PstI site found at 5061 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 9089
Illegal SpeI site found at 8430
Illegal PstI site found at 1240
Illegal PstI site found at 1714
Illegal PstI site found at 5061
Illegal NgoMIV site found at 1336
Illegal NgoMIV site found at 3654
Illegal NgoMIV site found at 4171
Illegal NgoMIV site found at 5512
Illegal NgoMIV site found at 5797
Illegal AgeI site found at 1430
Illegal AgeI site found at 3112 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1900
Illegal BsaI.rc site found at 3894
Illegal BsaI.rc site found at 7325
Illegal SapI site found at 6242
Illegal SapI.rc site found at 5361
Illegal SapI.rc site found at 5571
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