Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V015495 | pMESD22c7 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pMESD22c7 were constructed for subcloning nanobodies to express the megabodies Mbc7HopQ/Nb in the periplasm of E. coli. The includes the c7HopQ scaffold protein and nanobody beta-strain A (residues 1-14).
- Vector Name:
- pMESD22c7
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4434 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Uchanski T, Masiulis S, Fischer B, Kalichuk V
pMESD22c7 vector Vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
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
- Uchański T, Masiulis S, Fischer B, Kalichuk V, López-Sánchez U, Zarkadas E, Weckener M, Sente A, Ward P, Wohlkönig A, Zögg T, Remaut H, Naismith JH, Nury H, Vranken W, Aricescu AR, Pardon E, Steyaert J. Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. Nat Methods. 2021 Jan;18(1):60-68. doi: 10.1038/s41592-020-01001-6. Epub 2021 Jan 6. PMID: 33408403; PMCID: PMC7611088.
pMESD22c7 vector Sequence
LOCUS 62056_16750 4434 bp DNA circular SYN 07-JAN-2021
DEFINITION Cloning vector pMESD22c7, complete sequence.
ACCESSION MT338520
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4434)
AUTHORS Uchanski T, Masiulis S, Fischer B, Kalichuk V, Lopez-Sanchez U,
Zarkadas E, Weckener M, Sente A, Ward P, Wohlkoenig A, Zoegg T,
Remaut H, Naismith JH, Nury H, Vranken W, Aricescu AR, Pardon E,
Steyaert J.
TITLE Megabodies expand the nanobody toolkit for protein structure
determination by single-particle cryo-EM
JOURNAL Nat Methods 18 (1), 60-68 (2021)
REFERENCE 2 (bases 1 to 4434)
AUTHORS Steyaert J, Uchanski T, Pardon E.
TITLE Direct Submission
JOURNAL Submitted (14-APR-2020) VIB-VUB Center for Structural Biology, VIB,
Pleinlaan 2, Brussels 1050, Belgium
REFERENCE 3 (bases 1 to 4434)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods"; date: "2021"; volume: "18"; issue: "1"; pages: "60-68"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(14-APR-2020) VIB-VUB Center for Structural Biology, VIB, Pleinlaan
2, Brussels 1050, Belgium"
FEATURES Location/Qualifiers
source 1..4434
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 107..128
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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)."
misc_feature 225..281
/label=DsbA signal peptide
/note="DsbA signal peptide"
misc_feature 282..320
/label=nanobody beta-strain A (residues 1-14)
/note="nanobody beta-strain A (residues 1-14)"
misc_feature 321..1487
/label=c7HopQ scaffold protein
/note="c7HopQ scaffold protein"
misc_feature 1488..1490
/label=nanobody residue 17
/note="nanobody residue 17"
misc_feature 1491..1523
/label=multi cloning site
/note="multi cloning site"
CDS 1524..1541
/label=6xHis
/note="6xHis affinity tag"
misc_feature 1542..1553
/label=EPEA-tag
/note="EPEA-tag"
primer_bind complement(1563..1579)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 1792..2247
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2529..2633
/label=AmpR promoter
CDS 2634..3491
/label=AmpR
/note="beta-lactamase"
rep_origin 3665..4253
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"