质粒编号
质粒名称
规格
价格
V024572
pLV3-CMV-ITGA5(human)-FLAG-mCherry-mCherry-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-ITGA5(human)-FLAG-mCherry-mCherry-Puro
- 载体抗性:
- Ampicillin
- Refseq ID:
- NM_002205.5
- 载体长度:
- 11285 bp
- 载体类型:
- Protein expression
- 复制子:
- ori
- 宿主:
- Mammalian cells, Lentivirus
- 筛选标记:
- Puro;mCherry
- 启动子:
- CMV
- 载体标签:
- FLAG-mCherry
- 感受态:
- Stbl3
- 培养温度:
- 37℃
- 表达方法:
- Constiutive, Stable / Transient
- 描述:
- Homo sapiens integrin subunit alpha 5 (ITGA5), mRNA
pLV3-CMV-ITGA5(human)-FLAG-mCherry-mCherry-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-ITGA5(human)-FLAG-mCherry-mCherry-Puro 载体序列
LOCUS V024572 11285 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V024572
VERSION V024572
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 131..334
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 396..3542
/label="ITGA5(NM_002205)"
/note="ITGA5(NM_002205)"
/gene="ITGA5"
CDS 3549..3614
/codon_start=1
/product="three tandem FLAG® epitope tags, followed by an
enterokinase cleavage site"
/transl_table=1
/translation="DYKDHDGDYKDHDIDYKDDDDK"
/label="3xFLAG"
CDS 3618..4328
/codon_start=1
/note="mammalian codon-optimized"
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/transl_table=1
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK*"
/label="mCherry"
misc_feature 4368..4485
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
promoter 4534..5044
/note="human phosphoglycerate kinase 1 promoter"
/label="hPGK promoter"
CDS 5066..5665
/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"
LTR 5793..6026
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 6098..6219
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 6259..6394
/note="SV40 origin of replication"
/label="SV40 ori"
promoter complement(6415..6433)
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
primer_bind complement(6443..6459)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"
rep_origin 6601..7056
/direction=RIGHT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 7082..7186
/gene="bla"
/label="AmpR promoter"
CDS 7187..8047
/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"
rep_origin 8218..8806
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
protein_bind 9094..9115
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
promoter 9130..9160
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 9168..9184
/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"
primer_bind 9192..9208
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
promoter 9229..9247
/note="promoter for bacteriophage T3 RNA polymerase"
/label="T3 promoter"
promoter 9275..9501
/note="Rous sarcoma virus enhancer/promoter"
/label="RSV promoter"
LTR 9502..9682
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
/label="5' LTR (truncated)"
misc_feature 9729..9854
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 10347..10580
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 10765..10809
/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"