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:
- pRGK335
- 载体长度:
- 11051 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 宿主:
- Plants
- 载体来源:
- Engineer CB, Fitzsimmons KC, Schmuke JJ, Dotson SB, Kranz RG.
- 启动子:
- minimal CaMV 35S
pRGK335 载体载体图谱
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。
pRGK335 载体载体序列
LOCUS 40924_36963 11051 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pRGK335, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11051)
AUTHORS Engineer CB, Fitzsimmons KC, Schmuke JJ, Dotson SB, Kranz RG.
TITLE Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer
trap system in Arabidopsis
JOURNAL BMC Plant Biol. 5, 9 (2005)
PUBMED 15941484
REFERENCE 2 (bases 1 to 11051)
AUTHORS Engineer CB, Fitzsimmons KC, Schmuke J, Dotson S, Kranz RG.
TITLE Direct Submission
JOURNAL Submitted (01-SEP-2004) Biology, Washington University in St. Louis,
1 Brookings Drive, Biology 1137, St. Louis, MO 63130-4899, USA
REFERENCE 3 (bases 1 to 11051)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 11051)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "BMC Plant
Biol. 5, 9 (2005)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(01-SEP-2004) Biology, Washington University in St. Louis, 1
Brookings Drive, Biology 1137, St. Louis, MO 63130-4899, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11051
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(292..316)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
promoter 339..385
/label=minimal CaMV 35S promoter
/note="minimal 35S promoter from cauliflower mosaic virus"
CDS 486..926
/codon_start=1
/label=GAL4 DNA binding domain
/note="DNA binding domain of the GAL4 transcriptional
activator"
/translation="MKLLSSIEQACDICRLKKLKCSKKKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
CDS 930..1163
/codon_start=1
/label=VP16 AD
/note="transcriptional activation domain of herpes simplex
virus protein VP16 (Triezenberg et al., 1988; Cousens et
al., 1989)"
/translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
TPHDSAPYGALDTADFEFEQMFTDALGIDEYGG"
terminator 1170..1422
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 1670..1716
/label=minimal CaMV 35S promoter
/note="minimal 35S promoter from cauliflower mosaic virus"
CDS 1829..2377
/codon_start=1
/label=BlpR
/note="phosphinothricin acetyltransferase"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
protein_bind 2842..2936
/label=5X UAS
/note="five tandem copies of the 'ScaI site' 17-mer
CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS)
that efficiently binds yeast Gal4 (Webster et al., 1988;
Pfeiffer et al., 2010)"
misc_feature 2945..3041
/label=m35S
/note="m35S"
promoter 2997..3043
/label=minimal CaMV 35S promoter
/note="minimal 35S promoter from cauliflower mosaic virus"
CDS 3081..4730
/codon_start=1
/label=luciferase
/note="firefly luciferase"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
misc_feature 4776..5735
/label=TML
/note="TML"
protein_bind 5762..6032
/label=tetracycline response element
/note="contains seven copies of the tetracycline operator
tetO"
promoter 6073..6119
/label=minimal CaMV 35S promoter
/note="minimal 35S promoter from cauliflower mosaic virus"
misc_feature complement(6120..6144)
/label=LB T-DNA repeat
/note="left border repeat from octopine Ach5 T-DNA"
rep_origin complement(6310..7023)
/direction=LEFT
/label=oriV
/note="incP origin of replication"
CDS 8291..8479
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 8584..8724
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(8910..9498)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 10071..10859
/codon_start=1
/product="spectinomycin/streptomycin resistance"
/label=spectinomycin/streptomycin resistance
/note="Spc/Str"
/protein_id="AAY79159.1"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"