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pFLAG-CMV-2 vector (V011139)

Price Information

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V011139 pFLAG-CMV-2 In stock, instant shipping

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

Basic Vector Information

Vector Name:
pFLAG-CMV-2
Antibiotic Resistance:
Ampicillin
Length:
4679 bp
Type:
Mammalian Expression Vectors
Replication origin:
ori
Source/Author:
Sigma-Aldrich
Copy Number:
High copy number
Promoter:
CMV
5' Primer:
M13 fwd
3' Primer:
M13 rev

pFLAG-CMV-2 vector Map

Copy Sequence View Map
pFLAG-CMV-24679 bp600120018002400300036004200M13 fwdCMV enhancerCMV promoterATGFLAGhGH poly(A) signalSV40 promoterT7 promoterM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR 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.

pFLAG-CMV-2 vector Sequence

LOCUS       pFLAG-CMV-2.        4679 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector for expression of N-terminally FLAG-tagged proteins in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pFLAG-CMV-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4679)
  AUTHORS   Sigma-Aldrich
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4679)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             932..934
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             935..958
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     misc_feature    956..1022
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     polyA_signal    1020..1642
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        1671..2000
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(2039..2057)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2071..2087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2095..2111)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(2119..2149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2164..2185)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2473..3061)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3235..4092)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4093..4197)
                     /label=AmpR promoter
     rep_origin      4224..4679
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"