pTRXFUS vector (Cat. No.: V002519)

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Basic Information

Note: pTRXFUS (3.6 kb) is a bacterial expression vector for enhanced protein solubility. It features the lambda pL promoter driving expression of TrxA (thioredoxin) as a fusion partner, followed by an enterokinase site and a polylinker cloning site. The vector also includes the lambda N peptide for translational enhancement and an aspA transcription terminator. It carries M13 fwd and M13 rev priming sites for sequencing.

Name:: pTRXFUS

Antibiotic Resistance:: Ampicillin

Length:: 3585 bp

Type:: Fusion cloning vector

Replication origin:: ori

Source/Author:: LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM.

Growth Strain(s):: EPI400

Growth Temperature:: 37℃

$ 198.4

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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 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

Stoll VS, Manohar AV, Gillon W, MacFarlane EL, Hynes RC, Pai EF. A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization. Protein Sci. 1998 May;7(5):1147-55. doi: 10.1002/pro.5560070508. PMID: 9605319; PMCID: PMC2144001.

pTRXFUS vector (Cat. No.: V002519) Sequence

LOCUS       thioredoxin_gene        3585 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Fusion cloning vector pTRXFUS, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    thioredoxin gene fusion vector
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3585)
  AUTHORS   LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy 
            JM.
  TITLE     A thioredoxin gene fusion expression system that circumvents 
            inclusion body formation in the E. coli cytoplasm
  JOURNAL   Biotechnology (N.Y.) 11 (2), 187-193 (1993)
  PUBMED    7763371
REFERENCE   2  (bases 1 to 3585)
  AUTHORS   LaVallie ER.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-NOV-1994) Edward R. LaVallie, Genetics Institute, 87 
            CambridgePark Drive, Cambridge, MA 02140, USA
REFERENCE   3  (bases 1 to 3585)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3585)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biotechnology (N.Y.)"; date: "1993"; volume: "11"; issue: "2"; 
            pages: "187-193"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-NOV-1994) Edward R. LaVallie, Genetics Institute, 87 
            CambridgePark Drive, Cambridge, MA 02140, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3585
                     /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
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      2159..2187
                     /label=lambda pL promoter
                     /note="lambda pL promoter"
                     /regulatory_class="promoter"
     CDS             2416..2481
                     /label=lambda N peptide
                     /note="N-terminal RNA-binding domain from the lambda
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
     CDS             2724..3050
                     /label=TrxA
                     /note="E. coli thioredoxin"
     CDS             3066..3080
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     misc_feature    3081..3112
                     /label=polylinker cloning site
                     /note="polylinker cloning site"
     regulatory      3113..3179
                     /label=aspA transcription terminator
                     /note="aspA transcription terminator"
                     /regulatory_class="terminator"
     primer_bind     complement(3189..3205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"