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pNW33N vector (V004246)

Price Information

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V004246 pNW33N In stock, instant shipping

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

Basic Vector Information

AmpR fragment is non-functional - Chloramphenicol should be used as the resistance marker.

Vector Name:
pNW33N
Antibiotic Resistance:
Chloramphenicol
Length:
4217 bp
Type:
Geobacillus stearothermophilus Expression
Replication origin:
ori
Source/Author:
Mee EK, Welker NE.
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pNW33N vector Vector Map

pNW33N4217 bp600120018002400300036004200multiple cloning site from plasmid pUC19M13 revlac operatorlac promoterCAP binding siteChloramphenicol acetyltransferaserepBori

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

  • Nilasari D, Dover N, Rech S, Komives C. Expression of recombinant green fluorescent protein in Bacillus methanolicus. Biotechnol Prog. 2012 May-Jun;28(3):662-8.

pNW33N vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       V004246                 4217 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004246
VERSION     V004246
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 4217)
  AUTHORS   Mee EK, Welker NE.
  TITLE     Development of Genetic Tools to Analyze and Characterize Genes of
            the Thermophile Geobacillus stearothermophilus
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4217)
  AUTHORS   Mee EK, Welker NE.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-FEB-2003) Biochemistry, Molecular Biology, and
            Cellular Biology, Northwestern University, 2205 Tech Drive,
            Evanston, IL 60208, USA
REFERENCE   3  (bases 1 to 4217)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4217)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (14-FEB-2003) Biochemistry, Molecular Biology, and Cellular Biology,
            Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..51
                     /label="multiple cloning site from plasmid pUC19"
                     /note="multiple cloning site from plasmid pUC19"
     primer_bind     complement(69..85)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(93..109)
                     /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)."
     promoter        complement(117..147)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(162..183)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(316..963)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     CDS             complement(1073..2074)
                     /label="repB"
                     /note="RepB replication protein"
     rep_origin      complement(2766..3354)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    4217
                     /label="MCS"
                     /note="pUC18/19 multiple cloning site"