Note: pLKO.1 puro is a 3rd gen lentiviral backbone for cloning and expression of new shRNA sequences. The selection marker is puromycin.
基本信息
- 载体名称:
- pLKO.1 puro
- 载体抗性:
- Ampicillin
- 载体长度:
- 7032 bp
- 载体类型:
- Viral Expression & Packaging Vectors
- 复制子:
- ori
- 载体来源:
- Weinberg Lab / Addgene
- 筛选标记:
- Puromycin
- 拷贝数:
- High copy number
- 启动子:
- hPGK
- 感受态:
- JM108
- 培养温度:
- 37℃
产品信息
质粒编号:V010450
质粒名称:pLKO.1 puro
规格:5 μg (冻干粉)
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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。
pLKO.1 puro 质粒 (编号: V010450)序列
LOCUS Exported 7032 bp DNA circular SYN 11-SEP-2025
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7032)
AUTHORS hfytfiu
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7032)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7032
/mol_type="other DNA"
/organism="synthetic DNA construct"
source join(873..7032,1..872)
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 410..650
/label=U6 Promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 703..820
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 869..1379
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 1401..2000
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 2128..2361
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 2433..2554
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 2594..2729
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(2750..2768)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2778..2794)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 2936..3391
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3417..3521
/gene="bla"
/label=AmpR promoter
CDS 3522..4382
/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 4553..5141
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5429..5450
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5465..5495
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5503..5519
/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 5527..5543
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 5564..5582
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 5608..5836
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 5837..6017
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6064..6189
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 6682..6915
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."