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pEGFP-RhoA Biosensor vector (V006996)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V006996 pEGFP-RhoA Biosensor In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pEGFP-RhoA Biosensor
Antibiotic Resistance:
Kanamycin
Length:
6140 bp
Type:
Mammalian Expression
Replication origin:
ori
Selection Marker:
Neomycin (select with G418)
Promoter:
CMV
Cloning Method:
Restriction Enzyme
5' Primer:
CATGGACGAGCTGTACAAG

pEGFP-RhoA Biosensor vector Map

Copy Sequence View Map
pEGFP-RhoA Biosensor6140 bp30060090012001500180021002400270030003300360039004200450048005100540057006000CMV enhancerCMV promoterEGFPSV40 poly(A) signalf1 oriAmpR promoterSV40 promoterNeoR/KanRTK-pA-RHSV TK poly(A) signalori

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.

pEGFP-RhoA Biosensor vector Sequence

LOCUS       40924_17229        6140 bp DNA     circular SYN 13-MAY-2021
DEFINITION  C-terminus of Anillin in mammalian expression vector pEGFP-C1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  AUTHORS   Piekny AJ, Glotzer M
  TITLE     Anillin is a scaffold protein that links RhoA, actin, and myosin 
            during cytokinesis.
  JOURNAL   Curr Biol. 2008 Jan 8;18(1):30-6. Epub 2007 Dec 27.
  PUBMED    18158243
REFERENCE   2  (bases 1 to 6140)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6140)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Curr
            Biol."; date: "2008-01-8"; volume: "18(1)"; pages: "30-6. Epub 2007
            Dec 27"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             620..1336
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    2935..3056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(3063..3518)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3545..3649
                     /label=AmpR promoter
     promoter        3651..4008
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4043..4834
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5025..5044)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5069..5116
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5445..6033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"