Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000095 | pRNAT-U6.1/neo | 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
pRNAT-U6.1/Neo is designed for mammalian transfection. It carries a Neomycin resistance gene as the selectable marker, which can be used for establishing stable cell line. The GFP marker (coral GFP, cGFP) under CMV promoter control can be used to track the transfection efficiency. It uses U6 promoter for siRNA expression.
- Vector Name:
- pRNAT-U6.1/neo
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6380 bp
- Type:
- RNAi vectors
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Promoter:
- U6
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- pRNA-U6 Forward (TACGATACAAGGCTGTTAGAGAG)
- 3' Primer:
- pRNA Reverse (TAGAAGGCACAGTCGAGG)
- Fusion Tag:
- cGFP
pRNAT-U6.1/neo 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.
pRNAT-U6.1/neo vector Sequence
LOCUS 40924_37488 6380 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 6380)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6380)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6380
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 192..495
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 496..699
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 744..1460
/codon_start=1
/label=AcGFP1
/note="Aequorea coerulescens GFP"
/translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILL
EFVTAAGITHGMDELYK"
polyA_signal 1478..1599
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 1700..2128
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2142..2471
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2538..3329
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3506..3639
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3676..3692)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(3700..3716)
/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(3724..3754)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3769..3790)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4078..4663)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4837..5694)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5695..5799)
/label=AmpR promoter
promoter 6065..6083
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 6214..6380
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"