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:
- pN-PURO-PTP
- 载体抗性:
- Ampicillin
- 载体长度:
- 5650 bp
- 载体类型:
- Transfection vector
- 复制子:
- ori
- 载体来源:
- Schimanski B, Nguyen TN, Gunzl A.
pN-PURO-PTP 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。
pN-PURO-PTP 载体序列
LOCUS 40924_32963 5650 bp DNA circular SYN 18-DEC-2018 DEFINITION Transfection vector pN-PURO-PTP, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5650) AUTHORS Schimanski B, Nguyen TN, Gunzl A. TITLE Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination JOURNAL Eukaryotic Cell 4 (11), 1942-1950 (2005) PUBMED 16278461 REFERENCE 2 (bases 1 to 5650) AUTHORS Schimanski B, Nguyen TN, Gunzl A. TITLE Direct Submission JOURNAL Submitted (17-AUG-2005) Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030, USA REFERENCE 3 (bases 1 to 5650) TITLE Direct Submission REFERENCE 4 (bases 1 to 5650) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Eukaryotic Cell"; date: "2005"; volume: "4"; issue: "11"; pages: "1942-1950" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (17-AUG-2005) Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030, USA" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..5650 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 21..125 /label=AmpR promoter CDS 126..983 /label=AmpR /note="beta-lactamase" rep_origin 1157..1745 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 2033..2054 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 2069..2099 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2107..2123 /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 2131..2147 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 2168..2186 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_feature 2199..2204 /note="unique SacI restriction site for cloning the resistance marker cassette" misc_feature 2204..2702 /label=HSP70 genes 2 and 3 intergenic region /note="HSP70 genes 2 and 3 intergenic region" CDS 2703..3299 /label=PuroR /note="puromycin N-acetyltransferase" misc_feature 3315..4055 /label=beta-alpha tubulin intergenic region /note="beta-alpha tubulin intergenic region" misc_feature 4056..4061 /note="unique HindIII restriction site for cloning the resistance marker cassette and the PTP cassette" CDS 4526..4699 /label=ProtA /note="IgG-binding unit of Staphylococcus aureus protein A" CDS 4703..4873 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNDAQAPK" CDS 4910..4930 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" misc_feature 4973..4998 /note="multiple cloning site containing unique restriction sites for KpnI, ApaI, SnaBI and NotI for fusing the target protein to the PTP sequence" promoter complement(5007..5025) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5032..5048) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 5190..5645 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"