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。
- 载体名称:
- pHELLSGATE 8
- 载体抗性:
- Kanamycin
- 载体长度:
- 17485 bp
- 载体类型:
- Gateway Cloning Vectors
- 复制子:
- oriV
- 宿主:
- Plants
- 载体来源:
- Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM.
- 拷贝数:
- High copy number
- 启动子:
- CaMV 35S
pHELLSGATE 8 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。
pHELLSGATE 8 载体序列
LOCUS pHELLSGATE_8. 17485 bp DNA circular SYN 01-JAN-1980
DEFINITION High-throughput binary Gateway(R) destination vector for silencing
genes in plants using intron-containing hairpin RNA.
ACCESSION .
VERSION .
KEYWORDS pHELLSGATE 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17485)
AUTHORS Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM.
TITLE High-throughput vectors for efficient gene silencing in plants.
JOURNAL Funct. Plant Biol. 2002;29:1217-25.
REFERENCE 2 (bases 1 to 17485)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17485)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Funct.
Plant Biol."; date: "2002"; volume: "29"; pages: "1217-25"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17485
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(65..83)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(90..106)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 264..447
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 448..1266
/label=KanR
/note="fusion between an N-terminal peptide of nopaline
synthase and Tn5 aminoglycoside phosphotransferase"
terminator 1894..2146
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
misc_feature complement(2268..2292)
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
oriT 2846..2955
/label=oriT
/note="incP origin of transfer"
mobile_element 3015..3782
/label=IS1
/note="prokaryotic transposable element"
CDS 3782..4132
/label=traJ
/note="oriT-recognizing protein"
CDS 4407..5552
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
rep_origin complement(6915..7503)
/direction=LEFT
/label=ori
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 8600..9388
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase"
/label=aadA
/note="SmR"
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin complement(9993..10705)
/direction=LEFT
/label=oriV
/note="incP origin of replication"
misc_feature complement(11182..11206)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
protein_bind 11462..11483
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11498..11528
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11536..11552
/label=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 11560..11576
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 11594..11612
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
promoter 12672..13017
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
protein_bind 13027..13151
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(13583..13885)
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
protein_bind complement(14325..14449)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
intron 14476..15242
/label=PDK intron
/note="pyruvate orthophosphate dikinase intron from
Flaveria trinervia"
protein_bind 15285..15409
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(15749..16051)
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
protein_bind complement(16583..16707)
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
terminator 16716..17422
/label=OCS terminator
/note="octopine synthase terminator"
promoter complement(17463..17481)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"