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。
LentiGuide-zeo is a lentiviral CRISPR vector expressing a S. pyogenes chimeric RNA with a U6 promoter-driven customizable sgRNA for genome targeting and a zeocin resistance marker for mammalian cell selection. It enables precise Cas9-mediated editing and stable cell population enrichment via zeocin-based antibiotic selection.
- 载体名称:
- LentiGuide-zeo
- 载体抗性:
- Ampicillin
- 载体长度:
- 9964 bp
- 载体类型:
- Mammalian Expression, Lentiviral, CRISPR
- 筛选标记:
- Zeocin
- 拷贝数:
- High Copy
- 感受态:
- Stbl3
- 培养温度:
- 37℃
LentiGuide-zeo 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。
LentiGuide-zeo 载体序列
LOCUS Exported 9964 bp ds-DNA circular SYN 11-JUN-2020
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM recombinant plasmid
REFERENCE 1 (bases 1 to 9964)
AUTHORS Sascha
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9964
/organism="recombinant plasmid"
/mol_type="other DNA"
protein_bind 107..128
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 242..260
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 286..514
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 515..695
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 742..867
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1360..1593
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 2000..2240
/label=U6 Promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4130..4205
/label=gRNA scaffold
/note="guide RNA scaffold for the CRISPR/Cas9 system"
misc_feature 4261..4378
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 4517..5695
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 4748..5686
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind complement(5695..5713)
/label=Vector.REV
primer_bind 5699..5734
/label=Fragment.FOR (2)
CDS 5714..6088
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
primer_bind complement(6071..6103)
/label=Fragment.REV (2)
primer_bind 6089..6112
/label=Vector.FOR
misc_feature 6104..6692
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(6575..6586)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6764..6997
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7075..7196
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 7236..7371
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(7392..7410)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(7420..7436)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 7578..8033
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 8059..8163
/gene="bla"
/label=AmpR promoter
CDS 8164..9024
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 9195..9783
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"