35S:RUBY vector (V001191)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V001191	35S:RUBY	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

RUBY reporter converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. Tyrosine is first hydroxylated on the benzene ring, resulting in L-3,4-dihydroxyphenylalanine (L-DOPA). The reaction is catalyzed by the P450 oxygenase CYP76AD1. L-DOPA can be further oxidized into cyclo-DOPA by CYP76AD1. Alternatively, L-DOPA is catalyzed by L-DOPA 4,5-dioxygenase (DODA) into betalamic acid, which is subsequently condensed with cyclo-DOPA into betanidin. The condensation reaction does not require an enzyme. Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the colorful betalain. CYP76AD1, DODA, and Glucosyltransferase were organized into a single open reading frame to form the RUBY reporter and the three genes were linked by sequences that encode 2A peptides.

Vector Name:: 35S:RUBY

Antibiotic Resistance:: Spectinomycin

Length:: 14335 bp

Type:: Plant Expression

Replication origin:: ori

Host:: Plants

Promoter:: CaMV 35S (enhanced)

Cloning Method:: Gibson Cloning

5' Primer:: CCTGTCAAACACTGATAGTTTtgagacttttcaacaaagggt

3' Primer:: GCTTACTCAGTTAGGTCTAGCTTATCTTTAATCATATTCCATAGTCCA

Growth Strain(s):: Stbl3

Growth Temperature:: 37℃

35S:RUBY vector Map

Copy Sequence View 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.

References

He, Y., Zhang, T., Sun, H. et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res 7, 152 (2020). https://doi.org/10.1038/s41438-020-00390-1

35S:RUBY vector Sequence

LOCUS       V001191                14335 bp    DNA     circular SYN 25-DEC-2023
DEFINITION  Exported.
ACCESSION   V001191
VERSION     V001191
KEYWORDS    35S:RUBY
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 14335)
  AUTHORS   He Y, Zhang T, Sun H, Zhan H, Zhao Y
  TITLE     A reporter for noninvasively monitoring gene expression and plant
            transformation.
  JOURNAL   Hortic Res. 2020 Sep 19;7:152. doi: 10.1038/s41438-020-00390-1.
            eCollection 2020.
   PUBMED   33024566
REFERENCE   2  (bases 1 to 14335)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 14335)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Hortic
            Res."; date: "2020-09-19"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..14335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..708
                     /label="CaMV 35S promoter (enhanced)"
                     /note="cauliflower mosaic virus 35S promoter with a
                     duplicated enhancer region"
     CDS             717..2207
                     /gene="CYP76AD1"
                     /label="Cytochrome P450 76AD1"
                     /note="Cytochrome P450 76AD1 from Beta vulgaris.
                     Accession#: I3PFJ5"
     CDS             2217..2273
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2274..3098
                     /codon_start=1
                     /label="4,5-DOPA dioxygenase extradiol alpha 1"
                     /translation="MKMMNGEDANDQMIKESFFITHGNPILTVEDTHPLRPFFETWREK
                     IFSKKPKAILIISGHWETVKPTVNAVHINDTIHDFDDYPAAMYQFKYPAPGEPELARKV
                     EEILKKSGFETAETDQKRGLDHGAWVPLMLMYPEADIPVCQLSVQPHLDGTYHYNLGRA
                     LAPLKNDGVLIIGSGSATHPLDETPHYFDGVAPWAAAFDSWLRKALINGRFEEVNIYES
                     KAPNWKLAHPFPEHFYPLHVVLGAAGEKWKAELIHSSWDHGTLCHGSYKFTSA"
     CDS             3108..3164
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3165..4667
                     /codon_start=1
                     /label="cyclo-DOPA 5-O-glucosyltransferase"
                     /translation="MTAIKMNTNGEGETQHILMIPFMAQGHLRPFLELAMFLYKRSHVI
                     ITLLTTPLNAGFLRHLLHHHSYSSSGIRIVELPFNSTNHGLPPGIENTDKLTLPLVVSL
                     FHSTISLDPHLRDYISRHFSPARPPLCVIHDVFLGWVDQVAKDVGSTGVVFTTGGAYGT
                     SAYVSIWNDLPHQNYSDDQEFPLPGFPENHKFRRSQLHRFLRYADGSDDWSKYFQPQLR
                     QSMKSFGWLCNSVEEIETLGFSILRNYTKLPIWGIGPLIASPVQHSSSDNNSTGAEFVQ
                     WLSLKEPDSVLYISFGSQNTISPTQMMELAAGLESSEKPFLWVIRAPFGFDINEEMRPE
                     WLPEGFEERMKVKKQGKLVYKLGPQLEILNHESIGGFLTHCGWNSILESLREGVPMLGW
                     PLAAEQAYNLKYLEDEMGVAVELARGLEGEISKEKVKRIVEMILERNEGSKGWEMKNRA
                     VEMGKKLKDAVNEEKELKGSSVKAIDDFLDAVMQAKLEPSLQ"
     terminator      4675..4923
                     /label="HSP terminator"
                     /note="efficient transcription terminator from the
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya
                     et al., 2010)"
     promoter        5403..5586
                     /label="NOS promoter"
                     /note="nopaline synthase promoter"
     CDS             5638..6648
                     /label="HygR"
                     /note="aminoglycoside phosphotransferase from E. coli"
     terminator      6701..6953
                     /label="NOS terminator"
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(7528..7545)
                     /label="M13 Forward"
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     complement(7528..7544)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     complement(7537..7559)
                     /label="M13/pUC Forward"
                     /note="In lacZ gene"
     misc_feature    complement(7766..7790)
                     /label="LB T-DNA repeat"
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             8311..9099
                     /label="SmR"
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      9348..9936
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     10090..10107
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     misc_feature    complement(10122..10262)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     primer_bind     10348..10370
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     rep_origin      complement(10606..10800)
                     /direction=LEFT
                     /label="pVS1 oriV"
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(10869..11939)
                     /label="pVS1 RepA"
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(12371..12997)
                     /label="pVS1 StaA"
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"

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