质粒编号
质粒名称
规格
价格
V012734
pRGEB32
5 μg (冻干粉)
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学术研究使用，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。
Note:Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation.
- 载体名称:
- pRGEB32
- 载体抗性:
- Kanamycin
- 载体长度:
- 15888 bp
- 载体类型:
- Plant Vectors
- 复制子:
- ori
- 宿主:
- Plants
- 载体来源:
- Yinong Yang
- 筛选标记:
- Hygromycin
- 拷贝数:
- High copy number
- 启动子:
- CaMV 35S (enhanced)
- 感受态:
- JM108
- 培养温度:
- 37℃
pRGEB32 载体图谱
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。
pRGEB32 载体序列
LOCUS pRGEB32 15888 bp DNA circular SYN 29-DEC-2025
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15888)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 15888)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 15888)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..15888
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 26..49
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
terminator 71..323
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(332..348)
/label=M13 fwd
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 551..575
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 1876..2502
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
CDS 2940..4004
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 4073..4267
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 4611..4751
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(4937..5525)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5615..6406)
/label=KanR
/note="aminoglycoside phosphotransferase"
misc_feature 6831..6855
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal complement(6933..7107)
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(7151..8173)
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
promoter complement(8240..8916)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 9107..9128
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9143..9173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9181..9197
/label=lac repressor encoded by lacI binding site
/bound_moiety="lac repressor encoded by lacI"
/note="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)."
primer_bind 9205..9221
/label=M13 rev
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 9239..9619
/label=OsU3 promoter
/note="Oryza sativa (rice) snRNA U3 promoter"
misc_RNA 9636..9711
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
intron 10540..11501
/label=intron
/note="intron upstream of the coding sequence for the rice
polyubiquitin gene RUBQ2"
protein_bind 11512..11536
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 11581..11646
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 11653..11673
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 11698..15798
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 15799..15846
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"