pESC-URA 质粒 (编号: V010319)
基本信息
备注：The pESC-URA is a yeast episomal vector with a URA3 marker, for galactose-regulated expression and tagging of up to two genes.
- 载体名称
- pESC-URA
- 载体抗性
- Ampicillin
- 载体长度
- 6624 bp
- 载体类型
- Yeast Plasmids
- 复制子
- ori
- 宿主
- Yeast
- 载体来源
- Agilent Technologies
- 拷贝数
- High copy number
- 启动子
- GAL1,10
- 感受态
- DH10B
- 培养温度
- 37℃
质粒操作方法
质粒复溶方法
- 发货形式：质粒干粉，常温运输，收到后请于 -20 ℃ 保存，先转化提质粒再使用。
- 收到干粉后先 5000 rpm 离心 1 min，加入 20 μl ddH₂O 溶解。质粒复测浓度与标称值可能有差距，这通常来自干粉在管中的位置、复溶效率、测量偏差与管壁吸附，建议先转化提质粒后再使用。
- 取 1 支 100 μl 感受态细胞冰上解冻 10 min，加入 2 μl 质粒，冰浴 30 min，42 ℃ 热激 60 s（不要摇动），再冰浴 2 min。
- 加入 900 μl 无抗生素 LB 液体培养基，180 rpm、37 ℃ 振荡培养 45 min（30 ℃ 培养 1–1.5 h）。
- 6000 rpm 离心 5 min，保留约 100 μl 上清重悬菌体，涂布到对应抗性的 LB 平板。
- 平板倒置 37 ℃ 培养 14 h（30 ℃ 则 20 h）。菌落过多可稀释质粒后再转化；无菌落可增加至 10 μl 质粒。建议先转克隆感受态、重提质粒后再导入表达感受态。
- 挑取单菌落接种 LB 液体培养基，加入对应抗生素，220 rpm 振荡培养 14 h，按试剂盒说明提取质粒。
通用质粒转化方法
- 取100 μL感受态细胞置于冰上解冻10 min，加入2 μL质粒，冰浴30 min；随后42 ℃热激60 s，期间不要晃动，再冰浴2 min。
- 加入900 μL无抗LB液体培养基，37 ℃、180 rpm振荡培养45 min（30 ℃条件下则培养1–1.5 h）。
- 6000 rpm离心5 min，弃上清，仅保留100 μL上清重悬菌体沉淀，涂布于对应质粒抗性的LB平板上。
- 平板倒置培养，37 ℃培养14 h，或30 ℃培养20 h。
- 挑取单菌落至LB液体培养基中，加入对应抗生素，220 rpm振荡培养14 h；根据实验需求及质粒提取试剂盒说明书提取质粒。
参考文献
- Khulape SA, Maity HK, Pathak DC, Mohan CM, Dey S. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae. Acta Virol. 2015 Sep;59(3):240-6.
pESC-URA 质粒 (编号: V010319) 注释
LOCUS pESC-URA 6624 bp DNA circular SYN 20-JUL-2025
DEFINITION Cloning vector pESC-URA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6624)
AUTHORS Lu Q.
TITLE pESC-URA
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6624)
AUTHORS Zheng C-F.
TITLE Direct Submission
JOURNAL Submitted (06-MAY-1998) Technical Services, Stratagene, 11011 N.
Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE 3 (bases 1 to 6624)
AUTHORS Post K.
TITLE Direct Submission
JOURNAL Submitted (17-NOV-1999) Technical Services, Stratagene, 11011 N.
Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE 4 (bases 1 to 6624)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 6624)
TITLE Direct Submission
REFERENCE 6 (bases 1 to 6624)
TITLE Direct Submission
REFERENCE 7 (bases 1 to 6624)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(06-MAY-1998) Technical Services, Stratagene, 11011 N. Torrey Pines
Rd., La Jolla, CA 92037, USA"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(17-NOV-1999) Technical Services, Stratagene, 11011 N. Torrey Pines
Rd., La Jolla, CA 92037, USA"
COMMENT SGRef: number: 4; type: "Journal Article"
COMMENT SGRef: number: 5; type: "Journal Article"
COMMENT SGRef: number: 6; type: "Journal Article"
COMMENT On Nov 17, 1999 this sequence version replaced AF063585.1.
FEATURES Location/Qualifiers
source 1..6624
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 194..409
/label=URA3 promoter
CDS 410..1210
/codon_start=1
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
rep_origin complement(1344..1799)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
terminator complement(1878..2043)
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(2191..2214)
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
promoter 2261..2925
/label=GAL1,10 promoter
/note="divergent inducible promoter, regulated by Gal4"
promoter 2936..2954
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 2972..3001
/codon_start=1
/label=Myc
/note="Myc (human c-Myc proto-oncogene) epitope tag"
/translation="EQKLISEEDL"
terminator 3031..3220
/label=CYC1 terminator
/note="transcription terminator for CYC1"
rep_origin complement(3463..4051)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4225..5082)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5083..5187)
/label=AmpR promoter
rep_origin complement(5214..6556)
/direction=LEFT
/label=2u ori
/note="yeast 2u plasmid origin of replication"