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mito-PAGFP vector (V006707)

Price Information

Cat No. Plasmid Name Availability Add to cart
V006707 mito-PAGFP 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:
mito-PAGFP
Antibiotic Resistance:
Kanamycin
Length:
4759 bp
Type:
Mammalian Expression
Replication origin:
ori
Selection Marker:
Neomycin (select with G418)
Copy Number:
High Copy
Promoter:
CMV
Cloning Method:
Restriction Enzyme

mito-PAGFP vector Map

Copy Sequence View Map
mito-PAGFP4759 bp600120018002400300036004200SV40 promoterAmpR promoterf1 oriSV40 poly(A) signalPA-GFPCMV promoterCMV enhanceroriHSV TK poly(A) signalNeoR/KanR

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.

mito-PAGFP vector Sequence

LOCUS       40924_2004        4759 bp DNA     circular SYN 21-DEC-2021
DEFINITION  Expresses a mitochondrial matrix-targeted photoactivable GFP that 
            enables detection and quantification of organelle fusion in living 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4759)
  AUTHORS   Karbowski M, Arnoult D, Chen H, Chan DC, Smith CL, Youle RJ
  TITLE     Quantitation of mitochondrial dynamics by photolabeling of 
            individual organelles shows that mitochondrial fusion is blocked 
            during the Bax activation phase of apoptosis.
  JOURNAL   J Cell Biol. 2004 Feb 16. 164(4):493-9.
  PUBMED    14769861
REFERENCE   2  (bases 1 to 4759)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4759)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2004 Feb 16. 164(4):493-9."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(241..345)
                     /label=AmpR promoter
     rep_origin      372..827
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     polyA_signal    complement(834..955)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1081..1797)
                     /codon_start=1
                     /label=PA-GFP
                     /note="photoactivatable GFP variant (Patterson et al.,
                     2002)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(1934..2137)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(2138..2441)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2616..3204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(3533..3580)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(3815..4606)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(join(4641..4759,1..239))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"