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pDONR223_PIK3CA_p.H1047R vector (V006651)

Price Information

Cat No. Plasmid Name Availability Add to cart
V006651 pDONR223_PIK3CA_p.H1047R In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pDONR223_PIK3CA_p.H1047R
Antibiotic Resistance:
Spectinomycin
Length:
6000 bp
Type:
Gateway Entry vector
Replication origin:
ori
Copy Number:
High Copy
Promoter:
None
Cloning Method:
Gateway Cloning
5' Primer:
M13pUC-fwd
3' Primer:
M13-Rev

pDONR223_PIK3CA_p.H1047R vector Map

Copy Sequence View Map
pDONR223_PIK3CA_p.H1047R6000 bp60012001800240030003600420048005400L4440rrnB T2 terminatorrrnB T1 terminatorM13 fwdattL5Phosphatidylinositol 4,5-bisphosphate 3-kinasecatalytic subunit alpha isoformattL2T7 promoterM13 revSmRori

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.

pDONR223_PIK3CA_p.H1047R vector Sequence

LOCUS       V006651                 6000 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V006651
VERSION     V006651
KEYWORDS    pDONR223_PIK3CA_p.H1047R
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6000)
  AUTHORS   Berger AH, Brooks AN, Wu X, Shrestha Y, Chouinard C, Piccioni F,
            Bagul M, Kamburov A, Imielinski M, Hogstrom L, Zhu C, Yang X, Pantel
            S, Sakai R, Watson J, Kaplan N, Campbell JD, Singh S, Root DE,
            Narayan R, Natoli T, Lahr DL, Tirosh I, Tamayo P, Getz G, Wong B,
            Doench J, Subramanian A, Golub TR, Meyerson M, Boehm JS
  TITLE     High-throughput Phenotyping of Lung Cancer Somatic Mutations.
  JOURNAL   Cancer Cell. 2016 Aug 8;30(2):214-28. doi:
            10.1016/j.ccell.2016.06.022. Epub 2016 Jul 28.
   PUBMED   27478040
REFERENCE   2  (bases 1 to 6000)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6000)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.ccell.2016.06.022"; journalName: "Cancer Cell"; date:
            "2016-08-8- 8"; volume: "30"; issue: "2"; pages: "214-28"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(387..473)
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     537..553
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    570..665
                     /label="attL5"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             671..3874
                     /gene="PIK3CA"
                     /label="Phosphatidylinositol 4,5-bisphosphate 3-kinase
                     catalytic subunit alpha isoform"
                     /note="Phosphatidylinositol 4,5-bisphosphate 3-kinase
                     catalytic subunit alpha isoform from Homo sapiens.
                     Accession#: P42336"
     protein_bind    complement(3881..3978)
                     /label="attL2"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3996..4014)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4019..4035)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             4466..5254
                     /label="SmR"
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      5350..5938
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"