Note: pGADT7-GW is a Gateway-modified prey vector derived from standard pGADT7 for yeast two-hybrid assays. It carries GAL4 activation domain (AD) and attR recombination cassettes to enable fast ORF cloning via Gateway recombination. LEU2 marker allows yeast transformant selection, and ADH1 promoter drives fusion protein expression in yeast nuclei. This vector was built and applied in the TREX-2 interaction screening of the referenced Arabidopsis study.
基本信息
- 载体名称:
- pGADT7-GW
- 载体抗性:
- Ampicillin
- 载体长度:
- 9638 bp
- 载体类型:
- Hybridization
- 复制子:
- ori
- 宿主:
- Yeast
- 筛选标记:
- LEU2
- 启动子:
- ADH1(long)
- 感受态:
- DB3.1
- 培养温度:
- 37℃
产品信息
质粒编号:V013201
质粒名称:pGADT7-GW
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，供学术研究使用。
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。
参考文献
- Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Lu Q, Tang X, Tian G, Wang F, Liu K, Nguyen V, Kohalmi SE, Keller WA, Tsang EW, Harada JJ, Rothstein SJ, Cui Y. Plant J. 2010 Jan;61(2):259-70. doi: 10.1111/j.1365-313X.2009.04048.x. Epub 2009 Oct 16. 10.1111/j.1365-313X.2009.04048.x PubMed 19843313
pGADT7-GW 质粒 (编号: V013201)序列
LOCUS 62056_11205 9638 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9638)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9638
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 771..1476
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 1522..1542
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 1558..1899
/codon_start=1
/label=GAL4 activation domain
/note="activation domain of the GAL4 transcriptional
activator"
/translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
EDTPPNPKKE"
promoter 1905..1923
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1942..1968
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
protein_bind 1975..2099
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 2124..2154
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 2208..2864
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 3209..3511
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(3555..3679)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
terminator 4066..4253
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(4373..5464)
/codon_start=1
/label=LEU2
/note="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(5465..5870)
/label=LEU2 promoter
primer_bind complement(5912..5928)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5936..5952)
/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)."
promoter complement(5960..5990)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6005..6026)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
protein_bind 6081..6114
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
rep_origin complement(6465..7053)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7227..8084)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8085..8189)
/label=AmpR promoter
rep_origin 8471..9635
/label=2u ori
/note="yeast 2u plasmid origin of replication"