质粒编号
质粒名称
规格
价格
V001350
pTALEN_v2 (NG)
5 μg (冻干粉)
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:
- pTALEN_v2 (NG)
- 载体抗性:
- Chloramphenicol, Ampicillin
- 载体长度:
- 7961 bp
- 载体类型:
- Mammalian Expression
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- SV40
pTALEN_v2 (NG) 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。
pTALEN_v2 (NG) 载体序列
LOCUS 40924_42619 7961 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7961)
AUTHORS Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F
TITLE A transcription activator-like effector toolbox for genome
engineering.
JOURNAL Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431.
PUBMED 22222791
REFERENCE 2 (bases 1 to 7961)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7961)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nprot.2011"; journalName: "Nat Protoc"; date: "2012-01-5-
5"; volume: "7"; issue: "1"; pages: "171-92"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7961
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 86..674
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 828..845
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 944..960
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1416..1432
/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)."
enhancer 1459..1838
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1839..2042
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 2087..2105
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 2107..2116
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 2116..2181
/codon_start=1
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 2188..2208
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
promoter 2677..2707
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 2761..3417
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 3762..4064
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
CDS 4371..4958
/codon_start=1
/label=FokI cleavage domain
/note="nonspecific DNA cleavage domain of the FokI
endonuclease (Li et al., 1992)"
/translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
polyA_signal 4982..5093
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 5216..5545
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 5594..6616
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 6749..6882
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
CDS complement(6926..7783)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7784..7875)
/gene="bla"
/label=AmpR promoter
primer_bind 7917..7936
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 7942..7961
/label=pENTR-R
/note="pENTR vectors, reverse primer"