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pEGFP-rNFM vector (V001415)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V001415 pEGFP-rNFM 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-rNFM
Antibiotic Resistance:
Kanamycin
Length:
7464 bp
Type:
Mammalian Expression
Replication origin:
ori
Selection Marker:
Neomycin (select with G418)
Copy Number:
High Copy
Promoter:
CMV
Cloning Method:
Restriction Enzyme
5' Primer:
multiple primers
3' Primer:
multiple primers

pEGFP-rNFM vector Map

Copy Sequence View Map
pEGFP-rNFM7464 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200CMV enhancerCMV promoterEGFPNeurofilament medium polypeptideSV40 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-rNFM vector Sequence

LOCUS       V001415                 7464 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V001415
VERSION     V001415
KEYWORDS    pEGFP-rNFM
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7464)
  AUTHORS   Wang L, Ho CL, Sun D, Liem RK, Brown A
  TITLE     Rapid movement of axonal neurofilaments interrupted by prolonged
            pauses.
  JOURNAL   Nat Cell Biol. 2000 Mar;2(3):137-41.
   PUBMED   10707083
REFERENCE   2  (bases 1 to 7464)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7464)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Cell
            Biol."; date: "2000-03"; volume: "2(3)"; pages: "137-41"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             613..1329
                     /label="EGFP"
                     /note="enhanced GFP"
     CDS             1456..3939
                     /gene="Nefm"
                     /label="Neurofilament medium polypeptide"
                     /note="Neurofilament medium polypeptide from Rattus
                     norvegicus. Accession#: P12839"
     polyA_signal    4252..4373
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(4380..4835)
                     /direction=LEFT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4862..4966
                     /label="AmpR promoter"
     promoter        4968..5325
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     CDS             5360..6151
                     /label="NeoR/KanR"
                     /note="aminoglycoside phosphotransferase"
     primer_bind     complement(6342..6361)
                     /label="TK-pA-R"
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6386..6433
                     /label="HSV TK poly(A) signal"
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6762..7350
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"