质粒编号
质粒名称
规格
价格
V042468
pLV3-CMV-GLI3(human)-3xFLAG-CopGFP-Puro
100 μg (转染级)
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学术研究使用，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:
- pLV3-CMV-GLI3(human)-3xFLAG-CopGFP-Puro
- 载体抗性:
- Ampicillin
- Refseq ID:
- NM_000168.6
- 载体长度:
- 13030 bp
- 载体类型:
- Protein expression
- 复制子:
- ori
- 宿主:
- Mammalian cells, Lentivirus
- 筛选标记:
- Puro;CopGFP
- 拷贝数:
- High
- 启动子:
- CMV
- 5'引物:
- CMV-F
- 载体标签:
- 3xFLAG
- 感受态:
- Stbl3
- 培养温度:
- 37℃
- 表达方法:
- Constiutive, Stable / Transient
- 描述:
- Homo sapiens GLI family zinc finger 3 (GLI3), mRNA
pLV3-CMV-GLI3(human)-3xFLAG-CopGFP-Puro 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。
pLV3-CMV-GLI3(human)-3xFLAG-CopGFP-Puro 载体序列
LOCUS V042468 13030 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V042468
VERSION V042468
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
LTR 234..414
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
/label="5' LTR (truncated)"
misc_feature 458..583
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 1076..1309
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 1493..1537
/codon_start=1
/note="recognized by the 2H10 single-chain llama nanobody"
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/transl_table=1
/translation="KNEQELLELDKWASL"
/label="gp41 peptide"
misc_feature 1804..1921
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
promoter 2011..2214
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2317..7056
/label="GLI3(NM_000168)"
/note="GLI3(NM_000168)"
/gene="GLI3"
CDS 7063..7128
/codon_start=1
/product="three tandem FLAG® epitope tags, followed by an
enterokinase cleavage site"
/transl_table=1
/translation="DYKDHDGDYKDHDIDYKDDDDK"
/label="3xFLAG"
promoter 7172..7383
/note="core promoter for human elongation factor
EF-1α"
/label="EF-1α core promoter"
LTR 7396..7664
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
/label="5' LTR (truncated)"
regulatory 7693..7702
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
/label="Kozak sequence"
CDS 7723..8385
/codon_start=1
/product="green fluorescent protein 2 from Pontellina
plumata, also known as ppluGFP2 (Shagin et al., 2004)"
/transl_table=1
/translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
/label="CopGFP"
CDS 8455..8508
/codon_start=1
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/product="2A peptide from Thosea asigna virus capsid
protein"
/protein_id=""
/transl_table=1
/translation="EGRGSLLTCGDVEENPGP"
/label="T2A"
CDS 8509..9108
/codon_start=1
/gene="pac from Streptomyces alboniger"
/note="confers resistance to puromycin"
/product="puromycin N-acetyltransferase"
/transl_table=1
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
/label="PuroR"
misc_feature 9109..9697
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 9771..10004
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 10076..10197
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 10237..10372
/note="SV40 origin of replication"
/label="SV40 ori"
primer_bind complement(10405..10421)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 10429..10445
/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-β-D-thiogalactopyranoside (IPTG)."
/label="lac operator"
promoter complement(10453..10483)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 10498..10519
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(10807..11395)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(11566..12426)
/codon_start=1
/gene="bla"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/product="β-lactamase"
/transl_table=1
/translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
SLIKHW*"
/label="AmpR"
promoter complement(12427..12531)
/gene="bla"
/label="AmpR promoter"
primer_bind 13005..13021
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"