AbVec-hIgG1 vector (Cat. No.: V010129)
Note: AbVec-hIgG1 is a mammalian expression vector (AmpR) for human IgG1 heavy chain. It carries CMV enhancer/promoter, IgH signal peptide, hIgG1-CH constant region, Kozak sequence and SV40 polyA. Clone antibody VH regions into it to produce recombinant human IgG1 in 293/CHO cells.
- Name:
- AbVec-hIgG1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5741 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Di Niro R, Mesin L, Raki M, Zheng NY, Lund-Johansen F, Lundin KE, Charpilienne A, Poncet D, Wilson PC, Sollid LM.
- Copy Number:
- High copy number
- Promoter:
- SV40
- Growth Temperature:
- 37℃
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- [1] Dodev TS, Karagiannis P, Gilbert AE, et al. A tool kit for rapid cloning and expression of recombinant antibodies. Scientific Reports. 2014;4(1) DOI: 10.1038/srep05885
AbVec-hIgG1 vector (Cat. No.: V010129) Sequence
LOCUS Exported 5741 bp DNA circular SYN
DEFINITION Cloning vector AbVec-hIgG1, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5741)
AUTHORS Di Niro R, Mesin L, Raki M, Zheng NY, Lund-Johansen F, Lundin KE,
Charpilienne A, Poncet D, Wilson PC, Sollid LM.
TITLE Rapid Generation of Rotavirus-Specific Human Monoclonal Antibodies
from Small-Intestinal Mucosa
JOURNAL J. Immunol. 185 (9), 5377-5383 (2010)
PUBMED 20935207
REFERENCE 2 (bases 1 to 5741)
AUTHORS Wilson PC.
TITLE Antibody variable gene expression vector for human IgG1 heavy chain
JOURNAL Unpublished
REFERENCE 3 (bases 1 to 5741)
AUTHORS Wilson PC.
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2008) Medicine/Rheumatology, The University of
Chicago, BSLC/JFK Bldg. 924 East 57th St., Rm 414, Chicago, IL
60637, USA
REFERENCE 4 (bases 1 to 5741)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 5741)
TITLE Direct Submission
REFERENCE 6 (bases 1 to 5741)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Immunol."; date: "2010"; volume: "185"; issue: "9"; pages:
"5377-5383"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(18-NOV-2008) Medicine/Rheumatology, The University of Chicago,
BSLC/JFK Bldg. 924 East 57th St., Rm 414, Chicago, IL 60637, USA"
COMMENT SGRef: number: 4; type: "Journal Article"
COMMENT SGRef: number: 5; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5741
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 14..393
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 394..597
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 827..845
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
CDS 928..984
/label=IgH signal sequence
/note="signal sequence from the mouse immunoglobulin heavy
chain variable region"
CDS 994..1980
/label=hlgG1-CH
/note="Human IgG1 heavy chain constant region"
regulatory 1990..1999
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
polyA_signal 2003..2137
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter 2206..2563
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2583..2599)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 2812..3267
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3599..3703
/label=AmpR promoter
CDS 3704..4561
/label=AmpR
/note="beta-lactamase"
rep_origin 4735..5323
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5611..5632
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5647..5677
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5685..5701
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 5709..5725
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"