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pMID21 vector (V004730)

Price Information

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V004730 pMID21 In stock, instant shipping

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

Basic Vector Information

The pMID21 is derived from pCES119 where the Fab-display cassette has been replaced with a cassette nearly identical to that of DY3F63. The iii(stump) of pMID21 is flanked by two MluI sites. MluI digestion and religation removes iii(stump), allowing the expression of soluble Fab fragments in E. coli.

Vector Name:
pMID21
Antibiotic Resistance:
Ampicillin
Length:
5960 bp
Type:
Phagemid vector
Replication origin:
ori
Source/Author:
Hoet RM, Coehn EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Coolen-van Neer N, Nastri HG, Rondon IJ, Leeds J, Hufton SE, Huang L, Kashin I, De
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pMID21 vector Map

Copy Sequence View Map
pMID215960 bp60012001800240030003600420048005400AmpR promoterAmpRoriCAP binding sitelac promoterlac operatorM13 revencodes light chain signal sequence; antibody stufferencodes light chain antibody stufferhIg-kappa-CLheavy chain signalencodes heavy chain antibody stuffer6xHisMycM13 gene IIIM13 fwdf1 ori

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

  • Hoet RM, Cohen EH, Kent RB, et al. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol. 2005;23(3):344-348. doi:10.1038/nbt1067

  • Wassaf D, Kuang G, Kopacz K, et al. High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays. Anal Biochem. 2006;351(2):241-253. doi:10.1016/j.ab.2006.01.043

  • Kenniston JA, Faucette RR, Martik D, Comeau SR, Lindberg AP, Kopacz KJ, Conley GP, Chen J, Viswanathan M, Kastrapeli N, Cosic J, Mason S, DiLeo M, Abendroth J, Kuzmic P, Ladner RC, Edwards TE, TenHoor C, Adelman BA, Nixon AE, Sexton DJ. Inhibition of plasma kallikrein by a highly specific active site blocking antibody. J Biol Chem. 2014 Aug 22;289(34):23596-608. doi: 10.1074/jbc.M114.569061. Epub 2014 Jun 26. PMID: 24970892; PMCID: PMC4156074.

pMID21 vector Sequence

LOCUS       Exported                5960 bp DNA     circular SYN 22-OCT-2024
DEFINITION  Phagemid vector pMID21, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    pMID21 vector (V004730)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  AUTHORS   Hoet RM, Coehn EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem 
            L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Coolen-van Neer 
            N, Nastri HG, Rondon IJ, Leeds J, Hufton SE, Huang L, Kashin I, 
            Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, 
            Hoogenboom HR, Ladner RC.
  TITLE     Generation of high-affinity human antibodies by combining 
            donor-derived and synthetic complementarity-determining-region 
            diversity
  JOURNAL   Nat. Biotechnol. 23 (3), 344-348 (2005)
  PUBMED    15723048
REFERENCE   2  (bases 1 to 5960)
  AUTHORS   Ladner RC, Hoogenboom HR, Hoet RM, Cohen EH, Kashin I, Rondon IJ, 
            Rem L, Frans N, Schoonbroodt S, Kent RB, Rookey K, Hogan S.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-SEP-2004) Research, Dyax Corp, 300 Technology Square, 
            Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 5960)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5960)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 5960)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
            Biotechnol."; date: "2005"; volume: "23"; issue: "3"; pages: 
            "344-348"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (13-SEP-2004) Research, Dyax Corp, 300 Technology Square, Cambridge,
            MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        96..200
                     /label=AmpR promoter
     CDS             201..1058
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1232..1820
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2269..2325
                     /codon_start=1
                     
                     /note="encodes light chain signal sequence; antibody
                     stuffer"
                     /translation="VKKLLFAIPLVVPFYSHSA"
     misc_feature    2326..2352
                     /label=encodes light chain antibody stuffer
                     /note="encodes light chain antibody stuffer"
     CDS             2365..2682
                     /label=hIg-kappa-CL
                     /note="Human immunoglobulin kappa light chain constant
                     region"
     CDS             2726..2788
                     /codon_start=1
                     /label=heavy chain signal
                     /note="encodes heavy chain signal sequence; antibody
                     stuffer"
                     /translation="MKKLLFMIPLVVPFVAQPASA"
     misc_feature    2867..3766
                     /label=encodes heavy chain antibody stuffer
                     /note="encodes heavy chain antibody stuffer"
     CDS             4516..4533
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             4543..4572
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             4612..5070
                     /codon_start=1
                     /label=M13 gene III
                     /translation="SGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAI
                     DGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFV
                     FGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES"
     primer_bind     complement(5092..5108)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(5304..5759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"