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:
- pCX-OKS-2A
- 载体抗性:
- Ampicillin
- 载体长度:
- 8495 bp
- 载体类型:
- Mammalian Expression
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- CAG
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- GCGAGCCGCAGCCATTGCCTTTTA
- 3'引物:
- TTAGCCAGAAGTCAGATGCTC
pCX-OKS-2A 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。
pCX-OKS-2A 载体序列
LOCUS 40924_13930 8495 bp DNA circular SYN 13-MAY-2021 DEFINITION Non-integrating (episomal) expression of mouse Oct3/4, Klf4 and Sox2. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8495) AUTHORS Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S TITLE Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. JOURNAL Science. 2008 Oct 9. ():. PUBMED 18845712 REFERENCE 2 (bases 1 to 8495) TITLE Direct Submission REFERENCE 3 (bases 1 to 8495) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science. 2008 Oct 9. ():." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..8495 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 4..383 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 385..661 /label=chicken beta-actin promoter intron 662..1678 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1686..1705 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 1762..2817 /codon_start=1 /label=mPou5f1 /note="Mus musculus Oct-4 gene. Encodes a transcription factor containing a POU homeodomain that plays a key role in embryonic development and stem cell pluripotency." /translation="MAGHLASDFAFSPPPGGGDGSAGLEPGWVDSRTWLSFQGPPGGPG IGPGSEVLGISPCPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAGARVESNSEG TSSEPCADRPNAVKLEKVEPTPEESQDMKALQKELEQFAKLLKQKRITLGYTQADVGLT LGVLFGKVFSQTTICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQA RKRKRTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRS SIEYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGEAFPSV PVTALGSPMHSN" CDS 2842..2907 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" CDS 2911..4332 /codon_start=1 /label=mKlf4 /note="Mouse Kruppel-like factor (Klf4) gene. Belongs to the relatively large family of SP1-like transcription factors and is involved in the regulation of proliferation, differentiation, apoptosis and somatic cell reprogramming." /translation="MAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRL PPLPGRPYDLAATVATDLESGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTH QESVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAGGDPGVAASNTGGGLLYSR ESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKAS LTTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEA HLSAGPQLSNGHRPNTHDFPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPG PNYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAGCG KTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDR AFSRSDHLALHMKRHF" CDS 4357..4422 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" CDS 4447..5403 /codon_start=1 /label=mSox2 /note="Mus musculus transcription factor SOX-2 gene. Belongs to the SRY-related HMG-box (SOX) family of transcription factors, which is involved in the regulation of embryonic development and in the determination of cell fate" /translation="MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKR PMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHM KEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYA HMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGS PTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLP GAEVPEPAAPSRLHMAQHYQSGPVPGTAINGTLPLSHM" primer_bind complement(5516..5535) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 5581..5636 /label=beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(5635..5654) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(5997..6013) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(6021..6037) /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(6045..6075) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(6090..6111) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 6169..6365 /label=SV40 promoter /note="SV40 early promoter" polyA_signal 6371..6505 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6573..6590) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6744..7332) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7506..8363) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8364..8468) /label=AmpR promoter promoter 8495 /label=CAG /note="CMV early enhancer fused to modified chicken beta-actin promoter"