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pDONR223_PIK3CA_WT vector (V005924)

Price Information

Cat No. Plasmid Name Availability Add to cart
V005924 pDONR223_PIK3CA_WT 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_WT
Antibiotic Resistance:
Spectinomycin
Length:
5997 bp
Type:
Gateway Entry vector
Replication origin:
ori
Copy Number:
High Copy
Promoter:
T7
Cloning Method:
Gateway Cloning
5' Primer:
M13pUC-fwd
3' Primer:
M13-Rev

pDONR223_PIK3CA_WT vector Map

Copy Sequence View Map
pDONR223_PIK3CA_WT5997 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_WT vector Sequence

LOCUS       V005924                 5997 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V005924
VERSION     V005924
KEYWORDS    pDONR223_PIK3CA_WT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 5997)
  AUTHORS   Kim E, Ilic N, Shrestha Y, Zou L, Kamburov A, Zhu C, Yang X, Lubonja
            R, Tran N, Nguyen C, Lawrence MS, Piccioni F, Bagul M, Doench JG,
            Chouinard CR, Wu X, Hogstrom L, Natoli T, Tamayo P, Horn H, Corsello
            SM, Lage K, Root DE, Subramanian A, Golub TR, Getz G, Boehm JS, Hahn
            WC
  TITLE     Systematic Functional Interrogation of Rare Cancer Variants
            Identifies Oncogenic Alleles.
  JOURNAL   Cancer Discov. 2016 Jul;6(7):714-26. doi:
            10.1158/2159-8290.CD-16-0160. Epub 2016 May 4.
   PUBMED   27147599
REFERENCE   2  (bases 1 to 5997)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5997)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1158/2159-8290.CD-16-0160"; journalName: "Cancer Discov"; date:
            "2016-07"; volume: "6"; issue: "7"; pages: "714-26"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5997
                     /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(3878..3975)
                     /label="attL2"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3993..4011)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4016..4032)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             4463..5251
                     /label="SmR"
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      5347..5935
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"