Note:
基本信息
- 载体名称:
- pGBKT7
- 载体抗性:
- Kanamycin
- 载体长度:
- 7303 bp
- 载体类型:
- Yeast Plasmids
- 复制子:
- ori
- 载体来源:
- Clontech
- 筛选标记:
- Neomycin/G418(Geneticin)
- 拷贝数:
- High copy number
- 启动子:
- ADH1
- 3'引物:
- M13 rev
产品信息
质粒编号:V010264
质粒名称:pGBKT7
规格:5 μg (冻干粉)
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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。
pGBKT7 质粒 (编号: V010264)序列
LOCUS Exported 7303 bp DNA circular SYN 09-SEP-2012
DEFINITION Yeast two-hybrid ""bait"" vector for expressing proteins fused to
the GAL4 DNA-binding domain.
ACCESSION .
VERSION .
KEYWORDS pGBKT7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7303)
AUTHORS Clontech
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7303
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 30..734
/gene="S. cerevisiae ADH1"
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 762..1202
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/label=GAL4 DNA binding domain
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter 1213..1231
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1245..1247
/codon_start=1
/product="start codon for in vitro
transcription/translation"
/label=ATG
/translation="M"
CDS 1251..1280
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
misc_feature 1281..1333
/label=MCS
/note="multiple cloning site"
terminator 1338..1385
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
terminator 1412..1599
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for alcohol dehydrogenase
1"
primer_bind complement(1623..1639)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1647..1663
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
promoter complement(1671..1701)
/label=lac promoter
/note="promoter for the E. coli lac operon"
rep_origin complement(2025..2613)
/direction=LEFT
/label=ori
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 2942..2989
/label=HSV TK poly(A) signal
/note="herpesvirus thymidine kinase polyadenylation signal"
CDS complement(3221..4015)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin)"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(4016..4120)
/gene="bla"
/label=AmpR promoter
rep_origin 4147..5489
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 5748..6029
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
CDS 6030..6704
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(6807..7262)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"