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:
- NLSx3-CRY2PHR-p65
- 载体抗性:
- Ampicillin
- 载体长度:
- 8026 bp
- 载体类型:
- Mammalian Expression
- 复制子:
- ori
- 筛选标记:
- Neomycin (select with G418)
- 拷贝数:
- High Copy
- 启动子:
- CMV
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- CMV-F
- 3'引物:
- BGH Reverse
NLSx3-CRY2PHR-p65 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。
NLSx3-CRY2PHR-p65 载体序列
LOCUS 40924_2234 8026 bp DNA circular SYN 13-MAY-2021 DEFINITION Photoactivatable transcription system. Expression of activator probe, which includes the photolyase homology region of CRY2 (CRY2PHR) and the transcriptional activator domain.. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8026) AUTHORS Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M TITLE CRISPR-Cas9-based Photoactivatable Transcription System. JOURNAL Chem Biol. 2015 Feb 19;22(2):169-74. doi: 10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22. PUBMED 25619936 REFERENCE 2 (bases 1 to 8026) TITLE Direct Submission REFERENCE 3 (bases 1 to 8026) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1016/j.chembiol.2014.12.011"; journalName: "Chem Biol"; date: "2015-02-19- 19"; volume: "22"; issue: "2"; pages: "169-74" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..8026 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 908..917 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" CDS 923..943 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 959..979 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 1085..2578 /codon_start=1 /label=CRY2PHR /note="N-terminal photolyase homology region of Arabidopsis thaliana cryptochrome 2" /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA" CDS 2636..2662 /codon_start=1 /label=HA /note="HA (human influenza hemagglutinin) epitope tag" /translation="YPYDVPDYA" CDS 3092..3463 /codon_start=1 /label=RelA (p65) AD /note="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALLSQISS" CDS 3524..3565 /codon_start=1 /label=V5 tag /note="epitope tag from simian virus 5" /translation="GKPIPNPLLGLDST" CDS 3575..3592 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" polyA_signal 3621..3845 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3891..4319 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 4333..4662 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 4729..5520 /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" polyA_signal 5699..5832 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5869..5885) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(5893..5909) /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(5917..5947) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(5962..5983) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6100..6117) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6271..6859) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7033..7890) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7891..7995) /label=AmpR promoter