Human GSK3B ORF clone (NM_002093) (Cat. No.: V036244)

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Basic Information

Name:: pEnCMV-GSK3B(human)-EGFP-FLAG-SV40-Neo

Accession ID:: NM_002093.4

Antibiotic Resistance:: Kanamycin

Length:: 6930 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells

Selection Marker:: Neo/G418;EGFP

Promoter:: CMV

Fusion Tag:: EGFP-FLAG

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

Expression Method:: Transient

Transcript Definition:: Homo sapiens glycogen synthase kinase 3 beta (GSK3B), transcript variant 1, mRNA

Cat. No.: V036244 pEnCMV-GSK3B(human)-EGFP-FLAG-SV40-Neo

$ 298.8

In stock, 1-2 weeks for quality controls

Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

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.

Human GSK3B ORF clone (NM_002093) (Cat. No.: V036244) Sequence

LOCUS       V036244                 6930 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V036244
VERSION     V036244
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     primer_bind     166..182
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
     enhancer        343..722
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        723..926
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        952..970
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             1055..1771
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK"
                     /label="EGFP"
     CDS             1808..3106
                     /label="GSK3B(NM_002093)"
                     /note="GSK3B(NM_002093)"
                     /gene="GSK3B"
     CDS             3113..3178
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     primer_bind     complement(3187..3203)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     polyA_signal    3214..3836
                     /note="human growth hormone polyadenylation signal"
                     /label="hGH poly(A) signal"
     rep_origin      complement(3985..4573)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     polyA_signal    4902..4949
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     CDS             complement(5181..5975)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     promoter        complement(6010..6367)
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     promoter        complement(6369..6473)
                     /gene="bla"
                     /label="AmpR promoter"
     rep_origin      6500..6928
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"