Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001671 | pcDNA6.2-GWEmGFP-miR negative | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pcDNA6.2-GWEmGFP-miR negative
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5759 bp
- Type:
- Gateway System
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Promoter:
- CMV
- Cloning Method:
- Gateway
pcDNA6.2-GWEmGFP-miR negative vector Map
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.
pcDNA6.2-GWEmGFP-miR negative vector Sequence
LOCUS 40924_10276 5759 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5759)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5759)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5759
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 384..587
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 632..650
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 680..704
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 713..1429
/codon_start=1
/label=EmGFP
/note="Emerald GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
ncRNA 1492..1518
/label=5' miR-155
/note="sequence upstream of the precursor of mouse miR-155
microRNA (Uva et al., 2013)"
ncRNA 1582..1623
/label=3' miR-155
/note="sequence downstream of the precursor of mouse
miR-155 microRNA (Uva et al., 2013)"
protein_bind complement(1652..1676)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
polyA_signal 1762..1810
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter complement(1981..1999)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2004..2020)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 2088..2516
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2530..2859
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter 2907..2954
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 2973..3368
/codon_start=1
/label=BSD
/note="blasticidin S deaminase"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 3529..3662
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(3856..4444)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4543..5331)
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"