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pCMV6-Entry vector (V011173)

Price Information

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V011173 pCMV6-Entry In stock, instant shipping

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

Basic Vector Information

This is a mammalian expression with Myc-Flag, a dual tagging vector. It serves as an entry vector to transfer an ORF into a destination vector.

Vector Name:
pCMV6-Entry
Antibiotic Resistance:
Kanamycin
Length:
4919 bp
Type:
Mammalian Expression Vectors
Replication origin:
ori
Source/Author:
OriGene
Selection Marker:
Neomycin/G418(Geneticin)
Copy Number:
High copy number
Promoter:
SV40

pCMV6-Entry vector Map

Copy Sequence View Map
pCMV6-Entry4919 bp6001200180024003000360042004800M13 fwdCMV enhancerCMV promoterT7 promoterMCSFLAGM13 revhGH poly(A) signaloriHSV TK poly(A) signalNeoR/KanRSV40 promoterAmpR promoterf1 ori

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.

pCMV6-Entry vector Sequence

LOCUS       pCMV6-Entry.        4919 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian expression and dual tagging vector that serves as an entry
            vector to transfer an ORF into a destination vector using the 
            PrecisionShuttle(TM) system.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV6-Entry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4919)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4919)
  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..4919
                     /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"
     misc_feature    979..1087
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             1085..1114
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             1133..1156
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     primer_bind     complement(1176..1192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1203..1825
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(1974..2562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(2891..2938)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(3173..3964)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(3999..4356)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(4358..4462)
                     /label=AmpR promoter
     rep_origin      4489..4919
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"