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pDEST17-UbE2D3 vector (V006961)

Price Information

Cat No. Plasmid Name Availability Add to cart
V006961 pDEST17-UbE2D3 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:
pDEST17-UbE2D3
Antibiotic Resistance:
Ampicillin
Length:
3434 bp
Type:
Bacterial Expression
Replication origin:
ori
Copy Number:
Low Copy
Cloning Method:
Gateway Cloning
5' Primer:
T7

pDEST17-UbE2D3 vector Map

Copy Sequence View Map
pDEST17-UbE2D33434 bp6001200180024003000T7 promoterRBS6xHisT7 tag (gene 10 leader)Xpress(TM) tagattB1Ubiquitin-conjugating enzyme E2 D3attB2T7 terminatorf1 oriAmpR promoterAmpRoriL4440

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.

pDEST17-UbE2D3 vector Sequence

LOCUS       V006961                 3434 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V006961
VERSION     V006961
KEYWORDS    pDEST17-UbE2D3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 3434)
  AUTHORS   Jin J, Li X, Gygi SP, Harper JW
  TITLE     Dual E1 activation systems for ubiquitin differentially regulate E2
            enzyme charging.
  JOURNAL   Nature. 2007 Jun 28. 447(7148):1135-8.
   PUBMED   17597759
REFERENCE   2  (bases 1 to 3434)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3434)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
            2007 Jun 28. 447(7148):1135-8."
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /label="6xHis"
                     /note="6xHis affinity tag"
     CDS             114..146
                     /label="T7 tag (gene 10 leader)"
                     /note="leader peptide from bacteriophage T7 gene 10"
     CDS             150..173
                     /label="Xpress(TM) tag"
                     /note="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
     protein_bind    201..225
                     /label="attB1"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             246..686
                     /gene="UBE2D3"
                     /label="Ubiquitin-conjugating enzyme E2 D3"
                     /note="Ubiquitin-conjugating enzyme E2 D3 from Macaca
                     fascicularis. Accession#: Q4R5N4"
     protein_bind    complement(713..737)
                     /label="attB2"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      830..877
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     rep_origin      974..1429
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1455..1559
                     /label="AmpR promoter"
     CDS             1560..2417
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      2591..3179
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     3333..3350
                     /label="L4440"
                     /note="L4440 vector, forward primer"