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pBudCE4.1-EnCMV-MCS-EF1a-hrGFP vector (V013811)

Price Information

Cat No. Plasmid Name Availability Add to cart
V013811 pBudCE4.1-EnCMV-MCS-EF1a-hrGFP In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pBudCE4.1-EnCMV-MCS-EF1a-hrGFP
Antibiotic Resistance:
Bleomycin
Length:
5293 bp
Type:
Protein expression
Replication origin:
ori
Host:
Mammalian cells
Selection Marker:
Zeo
Promoter:
EF-1α
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pBudCE4.1-EnCMV-MCS-EF1a-hrGFP vector Map

Copy Sequence View Map
pBudCE4.1-EnCMV-MCS-EF1a-hrGFP5293 bp6001200180024003000360042004800CMV enhancerCMV promoterT7 promoterMyc6xHisSV40 poly(A) signalBleoREM7 promoterSV40 promoterEF-1-alpha promoterhrGFPV5 tag6xHisbGH 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.

pBudCE4.1-EnCMV-MCS-EF1a-hrGFP vector Sequence

LOCUS       62056_4240        5293 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5293)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             719..748
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             764..781
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    complement(800..933)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1066..1437)
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(1456..1503)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     promoter        complement(1551..1867)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        1889..3051
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             3071..3787
                     /codon_start=1
                     /label=hrGFP
                     /note="humanized Renilla green fluorescent protein"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     CDS             3825..3866
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     CDS             3876..3893
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    3922..4146
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(4278..4866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"