首页技术支持质粒载体

我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。

所有的产品都严格仅供科学研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。

确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)

开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。

由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。

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.

载体名称:
35S:RUBY
载体抗性:
Spectinomycin
载体长度:
14335 bp
载体类型:
Plant Expression
复制子:
ori
宿主:
Plants
启动子:
CaMV 35S (enhanced)
克隆方法:
Gibson Cloning
5'引物:
CCTGTCAAACACTGATAGTTTtgagacttttcaacaaagggt
3'引物:
GCTTACTCAGTTAGGTCTAGCTTATCTTTAATCATATTCCATAGTCCA
感受态:
Stbl3
培养温度:
37℃

35S:RUBY 载体图谱

35S:RUBY14335 bp7001400210028003500420049005600630070007700840091009800105001120011900126001330014000CaMV 35S promoter (enhanced)Cytochrome P450 76AD1P2A4,5-DOPA dioxygenase extradiol alpha 1P2Acyclo-DOPA 5-O-glucosyltransferaseHSP terminatorNOS promoterHygRNOS terminatorIn lacZ geneLB T-DNA repeatSmRoriL4440bompGEX 3'pVS1 oriVpVS1 RepApVS1 StaA

质粒操作方法

1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)

2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）

3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；

4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);

5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;

6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);

7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。

35S:RUBY 载体序列

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)"