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:
- pCEP4-M2L
- 载体抗性:
- Ampicillin
- 载体长度:
- 12852 bp
- 载体类型:
- Episomal/EBNA
- 复制子:
- ori
- 筛选标记:
- Hygromycin
- 拷贝数:
- Low Copy
- 启动子:
- CMV
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- IRES-F
- 3'引物:
- EBV_rev
pCEP4-M2L 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。
pCEP4-M2L 载体序列
LOCUS Exported 12852 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION used in the derivation of human iPS cells using non-integrating
episomal vectors; expresses cMyc and Lin28.
ACCESSION .
VERSION .
KEYWORDS pCEP4-M2L
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12852)
AUTHORS Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA
TITLE Human Induced Pluripotent Stem Cells Free of Vector and Transgene
Sequences
JOURNAL Science. 2009 Mar 26.
PUBMED 19325077
REFERENCE 2 (bases 1 to 12852)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science.
2009 Mar 26."
FEATURES Location/Qualifiers
source 1..12852
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 384..587
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 538..558
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 584..608
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 683..2002
/codon_start=1
/product="human c-Myc proto-oncogene"
/label=c-Myc
/translation="MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP
SEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTE
LLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPN
PARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDS
LLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGS
PSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNR
KCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKA
TAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA"
misc_feature 2069..2642
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 2092..2644
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(2236..2253)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 2463..2482
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
polyA_signal 3338..3472
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3362..3381)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 3416..3435
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(3615..3637)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin 4195..5984
/label=oriP
/note="Epstein-Barr virus oriP replication origin (Yates et
al., 2000)"
CDS complement(6286..8211)
/codon_start=1
/product="Epstein-Barr nuclear antigen 1, also known as
EBNA-1"
/label=EBNA1
/note="crucial for latent viral infection, and for episomal
amplification of vectors containing the oriP origin (Okita
et al., 2013)"
/translation="MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGR
GRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAG
AGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGA
GGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGG
GAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAG
GGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRG
GSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPP
PGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGR
RKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSL
YNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLK
DAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE
GEEGQE"
primer_bind complement(8646..8664)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8732..8836
/gene="bla"
/label=AmpR promoter
CDS 8837..9697
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(9055..9074)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9868..10456
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10357..10376
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 10887..11032
/label=HSV TK promoter
/note="herpes simplex virus thymidine kinase promoter"
CDS 11075..12112
/codon_start=1
/product="hygromycin B phosphotransferase"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
MGEAN"
primer_bind complement(12110..12129)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 12154..12202
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"