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pNCO113-hisactophilin vector (V004342)

Price Information

Cat No. Plasmid Name Availability Add to cart
V004342 pNCO113-hisactophilin In stock, 1 week for quality controls

Buy one, get one free!

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

Basic Vector Information

Vector Name:
pNCO113-hisactophilin
Antibiotic Resistance:
Ampicillin
Length:
3776 bp
Type:
Expression vector
Replication origin:
ori
Source/Author:
Kay CW, Schleicher E, Kuppig A, Hofner H, Rudiger W, Schleicher M, Fischer M, Bacher A, Weber S, Richter G.
Promoter:
T5

pNCO113-hisactophilin vector Map

Copy Sequence View Map
pNCO113-hisactophilin3776 bp60012001800240030003600T5 promoterlac operatorRBSHisactophilin-1lambda t0 terminatorrrnB T1 terminatorbomoriAmpRAmpR promoter

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.

pNCO113-hisactophilin vector Sequence

LOCUS       V004342                 3776 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004342
VERSION     V004342
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 3776)
  AUTHORS   Kay CW, Schleicher E, Kuppig A, Hofner H, Rudiger W, Schleicher M,
            Fischer M, Bacher A, Weber S, Richter G.
  TITLE     Blue light perception in plants. Detection and characterization of a
            light-induced neutral flavin radical in a C450A mutant of
            phototropin
  JOURNAL   J. Biol. Chem. 278 (13), 10973-10982 (2003)
   PUBMED   12525505
REFERENCE   2  (bases 1 to 3776)
  AUTHORS   Fischer MJ, Bacher A.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-SEP-2002) Organic Chemistry and Biochemistry,
            Technical University Munich, Lichtenbergstr. 4, Garching 85747,
            Germany
REFERENCE   3  (bases 1 to 3776)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3776)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Biol.
            Chem."; date: "2003"; volume: "278"; issue: "13"; pages:
            "10973-10982"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (09-SEP-2002) Organic Chemistry and Biochemistry, Technical
            University Munich, Lichtenbergstr. 4, Garching 85747, Germany"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..54
                     /label="T5 promoter"
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    62..78
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and
                     Leibler, 2000)"
     CDS             115..468
                     /gene="hatA"
                     /label="Hisactophilin-1"
                     /note="Hisactophilin-1 from Dictyostelium discoideum.
                     Accession#: P13231"
     terminator      518..612
                     /label="lambda t0 terminator"
                     /note="transcription terminator from phage lambda"
     terminator      1384..1470
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     misc_feature    1626..1766
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(1952..2540)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(2714..3571)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(3572..3676)
                     /label="AmpR promoter"