pCMV-MIR vector (Cat. No.: V011248)

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Basic Information

Note: The pCMV-MIR vector is designed for the expression of microRNA (miRNA) precursors under the control of the CMV promoter. It includes a GFP reporter for monitoring transfection efficiency and a neomycin resistance gene for selection purposes.

Name:: pCMV-MIR

Antibiotic Resistance:: Kanamycin

Length:: 6219 bp

Type:: Mammalian Expression Vectors

Replication origin:: ori

Source/Author:: OriGene

Selection Marker:: Neomycin/G418(Geneticin)

Copy Number:: High copy number

Promoter:: SV40

Growth Strain(s):: DH10b

Growth Temperature:: 37℃

$ 198.5

In stock, instant shipping

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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.

References

McGrath J, Kane LE, Maher SG. The Influence of MicroRNA-31 on Oxidative Stress and Radiosensitivity in Pancreatic Ductal Adenocarcinoma. Cells. 2022;11(15):2294. Published 2022 Jul 25. doi:10.3390/cells11152294

pCMV-MIR vector (Cat. No.: V011248) Sequence

LOCUS       40924_11700        6219 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector for expressing microRNA (miRNA) precursors using the CMV 
            promoter, with a GFP reporter for transfection and a neomycin 
            resistance marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6219)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6219)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Inserts are cloned between the SgfI (AsiSI) and MluI sites.
FEATURES             Location/Qualifiers
     source          1..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1084..1113
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             1132..1155
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     primer_bind     complement(1175..1191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1202..1824
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(1973..2561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(2890..2937)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(3162..3857)
                     /codon_start=1
                     /label=TurboGFP
                     /note="improved green fluorescent protein from Pontellina 
                     plumata (Evdokimov et al., 2006)"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     misc_feature    complement(3858..4437)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             complement(4462..5253)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(5288..5645)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(5647..5751)
                     /label=AmpR promoter
     rep_origin      5778..6206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"