Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000005 | pGreenIIM RPS5A-mDII-ntdTomato/RPS5A-DII-n3Venus | 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
Kanamycin low (25 ug/ml)
- Vector Name:
- pGreenIIM RPS5A-mDII-ntdTomato/RPS5A-DII-n3Venus
- Antibiotic Resistance:
- Kanamycin
- Length:
- 12920 bp
- Type:
- Plant Expression
- Replication origin:
- pSa ori
- Host:
- Plants
- Selection Marker:
- Methothrexate
- Copy Number:
- Low Copy
- Promoter:
- CaMV 35S
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- EGFP_N_primer
pGreenIIM RPS5A-mDII-ntdTomato/RPS5A-DII-n3Venus vector 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.
pGreenIIM RPS5A-mDII-ntdTomato/RPS5A-DII-n3Venus vector Sequence
LOCUS 40924_22627 12920 bp DNA circular SYN 13-MAY-2021
DEFINITION R2D2: Expresses a fusion of auxin-sensitive DII to nuclear 3xVenus
and auxin-insensitive mDII to nuclear tdTomato, both from the
Arabidopsis RPS5A promoter.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12920)
AUTHORS Liao CY, Smet W, Brunoud G, Yoshida S, Vernoux T, Weijers D
TITLE Reporters for sensitive and quantitative measurement of auxin
response.
JOURNAL Nat Methods. 2015 Mar;12(3):207-10. doi: 10.1038/nmeth.3279. Epub
2015 Feb 2.
PUBMED 25643149
REFERENCE 2 (bases 1 to 12920)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12920)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nmeth.3279"; journalName: "Nat Methods"; date: "2015-03";
volume: "12"; issue: "3"; pages: "207-10"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12920
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(1721..1739)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1752..1776
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 1912..1932
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1933..3360
/codon_start=1
/label=tdTomato
/note="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
promoter complement(5352..5370)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5383..5407
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 5543..5563
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 5594..6310
/codon_start=1
/label=Venus
/note="yellow fluorescent protein (YFP) with fast and
efficient maturation (Nagai et al., 2002)"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 6329..6349
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 6380..7096
/codon_start=1
/product="yellow fluorescent protein (YFP) with fast and
efficient maturation (Nagai et al., 2002)"
/label=Venus
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 7130..7849
/codon_start=1
/product="yellow fluorescent protein (YFP) with fast and
efficient maturation (Nagai et al., 2002)"
/label=Venus
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
promoter complement(8133..8151)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8172..8188)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8172..8188)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8185..8207)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8196..8212
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(8220..8250)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8265..8286)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8403..8420)
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 8525..8549
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
rep_origin complement(8640..9228)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9402..10214)
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin 10505..10940
/label=pSa ori
/note="origin of replication from bacterial plasmid pSa"
misc_feature 11067..11089
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA
(truncated)"
polyA_signal 11097..11273
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(11672..12232)
/codon_start=1
/label=DHFR
/note="mouse dihydrofolate reductase"
/translation="MVRPLNCIVAVSQNMGIGKNGDRPWPPLRNEFKYFQRMTTTSSVE
GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
PGVLSEVQEEKGIKYKFEVYEKKD"
promoter complement(12265..12610)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
primer_bind 12828..12844
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 12854..12872
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"