35S:RUBY vector (V001191)

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V001191 35S:RUBY In stock (lyophilized plasmid)

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Two vials of lyophilized plasmid will be delivered, each vial 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
      • Promoter:
      • CaMV 35S
      • Cloning Method:
      • Gibson Cloning
      • 5' Primer:
      • CCTGTCAAACACTGATAGTTTtgagacttttcaacaaagggt
      • 3' Primer:
      • GCTTACTCAGTTAGGTCTAGCTTATCTTTAATCATATTCCATAGTCCA
      • Growth Strain(s):
      • MC1061
      • Growth Temperature:
      • 37℃

35S:RUBY vector Vector Map

35S:RUBY14335 bp7001400210028003500420049005600630070007700840091009800105001120011900126001330014000CaMV 35S promoter (enhanced)CYP76AD1P2A4,5-DOPA dioxygenase extradiol alpha 1P2Acyclo-DOPA 5-O-glucosyltransferaseNOS promoterHygRNOS terminatorM13 ForwardLB T-DNA repeatSmRoriL4440bompGEX 3'pVS1 oriVpVS1 RepApVS1 StaARB T-DNA repeat

Plasmid Resuspension 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 ℃.

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

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               14335 bp DNA     circular SYN 25-DEC-2023
DEFINITION  RUBY under the control of CaMV 35S promoter/marker for 
            transformation.
ACCESSION   .
VERSION     .
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"; pages: "
            10.1038/s41438-020-00390-1. eCollection 2020"
COMMENT     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"
     primer_bind     645..665
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             717..2213
                     /codon_start=1
                     /label=CYP76AD1
                     /translation="MDHATLAMILAIWFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTFEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGS"
     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"
     promoter        5371..5550
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        5403..5586
                     /function="NOS promoter"
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             5638..6663
                     /codon_start=1
                     /product="aminoglycoside O-phosphotransferase APH(4)-Ia 
                     from Salmonella enterica"
                     /label=HygR
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDIGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPGSW
                     R"
     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    7766..7790
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             8311..9102
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy,
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      9348..9936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9837..9856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10090..10107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    10122..10262
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(10188..10207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     10348..10370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      10606..10800
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(10866..11939)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(12368..12997)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    join(14328..14335,1..17)
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"