pMESy4 vector (V004763)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V004763	pMESy4	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

Vector Name:: pMESy4

Antibiotic Resistance:: Ampicillin

Length:: 4513 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Pardon E, Steyaert J.

pMESy4 vector Map

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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

Pardon E, Laeremans T, Triest S, Rasmussen SG, Wohlkönig A, Ruf A, Muyldermans S, Hol WG, Kobilka BK, Steyaert J. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014 Mar;9(3):674-93. doi: 10.1038/nprot.2014.039. Epub 2014 Feb 27. PMID: 24577359; PMCID: PMC4297639.

pMESy4 vector Sequence

LOCUS       Exported                4513 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Cloning vector pMESy4, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4513)
  AUTHORS   Pardon E, Steyaert J.
  TITLE     A general protocol for the generation of Nanobodies for structural 
            biology
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4513)
  AUTHORS   Pardon E, Steyaert J.
  TITLE     Direct Submission
  JOURNAL   Submitted (16-JUL-2013) Structural Biology Brussels, Vrije 
            Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium
REFERENCE   3  (bases 1 to 4513)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4513)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 4513)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (16-JUL-2013) Structural Biology Brussels, Vrije Universiteit
            Brussel, Pleinlaan 2, Brussels 1050, Belgium"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..4513
                     /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)."
     regulatory      211..215
                     /regulatory_class="ribosome_binding_site"
     sig_peptide     225..290
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     CDS             340..357
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    358..369
                     /label=CaptureSelect C-tag
                     /note="CaptureSelect C-tag"
     misc_feature    373..390
                     /label=HA-tag
                     /note="HA-tag"
     gene            403..1636
                     /gene="M13 geneIII"
                     /label=M13 geneIII
     primer_bind     complement(1642..1658)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      1871..2326
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2608..2712
                     /label=AmpR promoter
     CDS             2713..3570
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      3744..4332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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