pMESP23E2 (2 weeks) vector (Cat. No.: V016519)

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Basic Information

Note: This vector includes a cYgjK scaffold protein. The GenBank accession number is MT338521.

Name:: pMESP23E2 (2 weeks)

Antibiotic Resistance:: Ampicillin

Length:: 5601 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Uchanski T, Masiulis S, Fischer B, Kalichuk V

Copy Number:: High copy number

Growth Temperature:: 37℃

$ 342.5

In stock, 1 week for quality controls

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

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.

pMESP23E2 (2 weeks) vector (Cat. No.: V016519) Sequence

LOCUS       62056_16755        5601 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Cloning vector pMESP23E2, complete sequence.
ACCESSION   MT338521
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  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 5601)
  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 5601)
  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"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5601
                     /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)."
     sig_peptide     225..290
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     misc_feature    291..326
                     /label=nanobody beta-strain A (residues 1-13)
                     /note="nanobody beta-strain A (residues 1-13)"
     misc_feature    330..2654
                     /label=cYgjK scaffold protein
                     /note="cYgjK scaffold protein"
     misc_feature    2658..2690
                     /label=multi cloning site
                     /note="multi cloning site"
     CDS             2691..2708
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    2709..2720
                     /label=EPEA-tag
                     /note="EPEA-tag"
     primer_bind     complement(2730..2746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2959..3414
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3696..3800
                     /label=AmpR promoter
     CDS             3801..4658
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4832..5420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"