pECFP-Peroxi vector (V001407)

Price Information

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

Basic Vector Information

pECFP-Peroxi encodes a fusion of the enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP) and the peroximal targeting signal 1 (PTS1). The PTS1 sequence is fused to the 3' end of ECFP and encodes the tripeptide SKL, which targets the fusion protein to the matrix of peroxisomes (1–4). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission variants (5, 6). The other five substitutions enhance the brightness and solubility of the protein, primarily due to improved protein folding properties and efficiency of chromophore formation (5, 7, 8).In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (9, 10). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (11). These changes increase the translational efficiency of the ECFP-PTS1 mRNA and consequently the expression of ECFP-PTS1 in mammalian and plant cells.The expression of ECFP-PTS1 is driven by the immediate early promoter of CMV (PCMV IE). pECFPPeroxi contains an MCS at the 5' end of the ECFP-PTS1 sequence. Genes of interest cloned into the MCS will be expressed as fusions to the N-terminus of ECFP-PTS1 if they are in the same reading frame as ECFP-PTS1 and there are no intervening stop codons.The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.Expression of ECFP-PTS1 can be used simply for the labeling of peroxisomes. The MCS at the 5' end of ECFP-PTS1 may be used to fuse genes of interest to ECFP-PTS1. The inserted gene should include the initiation ATG codon and be in frame with ECFP-PTS1. The recombinant ECFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (12). ECFP can be used for double-or triple-labeling experiments together with EYFP and DsRed using standard fluorescence microscopy and the appropriate filter sets.

pECFP-Peroxi vector Map

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.

pECFP-Peroxi vector Sequence

LOCUS       40924_16750        4740 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4740)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4740)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4740
                     /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"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     CDS             679..1395
                     /codon_start=1
                     /label=ECFP
                     /note="enhanced CFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1528..1649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1656..2111)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2138..2242
                     /label=AmpR promoter
     promoter        2244..2601
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2636..3427
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3662..3709
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4038..4626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"