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pCMV-Tag 4A vector (V011235)

Price Information

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V011235 pCMV-Tag 4A In stock, instant shipping

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

Basic Vector Information

The pCMV-Tag 4A is a mammalian expression vector for tagging proteins with a C-terminal FLAG epitope. Using pCMV-Tag 4B or pCMV-Tag 4C for other reading frames.

Vector Name:
pCMV-Tag 4A
Antibiotic Resistance:
Kanamycin
Length:
4319 bp
Type:
Mammalian Expression Vectors
Replication origin:
ori
Source/Author:
Agilent Technologies
Selection Marker:
Neomycin/G418(Geneticin)
Copy Number:
High copy number
Promoter:
CMV
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pCMV-Tag 4A vector Map

Copy Sequence View Map
pCMV-Tag 4A4319 bp600120018002400300036004200CMV enhancerCMV promoterT3 promoterKS primerFLAGstop codonsT7 promoterSV40 poly(A) signalf1 oriAmpR promoterSV40 promoterNeoR/KanRHSV 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.

References

  • Jing H, Zhang X, Wisner SA, Chen X, Spiegelman NA, Linder ME, Lin H. SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a. Elife. 2017 Dec 14;6:e32436.

pCMV-Tag 4A vector Sequence

LOCUS       pCMV-Tag_4A.        4319 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian expression vector for tagging proteins with a C-terminal 
            FLAG epitope. For other reading frames, use pCMV-Tag 4B or pCMV-Tag 
            4C.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-Tag 4A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4319)
  AUTHORS   Agilent Technologies
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4319)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     The cloned gene must include a Kozak sequence and start codon.
FEATURES             Location/Qualifiers
     source          1..4319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        620..638
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(725..741)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             744..767
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     misc_feature    783..793
                     /label=stop codons
                     /note="stop codons"
                     /note="stop codons in all three reading frames"
     promoter        complement(821..839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1113..1234
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1241..1696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1723..1825
                     /label=AmpR promoter
     promoter        1829..2186
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2221..3012
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    3247..3294
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3623..4211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"