基本信息
- 载体名称
- pLV3-CMV-LARP1(human)-HA-EF1a-Puro
- 载体抗性
- Ampicillin
- Refseq ID
- NM_033551.3
- 载体长度
- 10757 bp
- 载体类型
- Protein expression
- 复制子
- ori
- 宿主
- Mammalian cells, Lentivirus
- 筛选标记
- Puro
- 启动子
- CMV
- 载体标签
- HA
- 感受态
- Stbl3
- 培养温度
- 37℃
- 表达方法
- Constiutive, Stable / Transient
- 描述
- Homo sapiens La ribonucleoprotein 1, translational regulator (LARP1), transcript variant 2, mRNA
质粒操作方法
质粒复溶方法
- 发货形式：质粒干粉，常温运输，收到后请于 -20 ℃ 保存，先转化提质粒再使用。
- 收到干粉后先 5000 rpm 离心 1 min，加入 20 μl ddH₂O 溶解。质粒复测浓度与标称值可能有差距，这通常来自干粉在管中的位置、复溶效率、测量偏差与管壁吸附，建议先转化提质粒后再使用。
- 取 1 支 100 μl 感受态细胞冰上解冻 10 min，加入 2 μl 质粒，冰浴 30 min，42 ℃ 热激 60 s（不要摇动），再冰浴 2 min。
- 加入 900 μl 无抗生素 LB 液体培养基，180 rpm、37 ℃ 振荡培养 45 min（30 ℃ 培养 1–1.5 h）。
- 6000 rpm 离心 5 min，保留约 100 μl 上清重悬菌体，涂布到对应抗性的 LB 平板。
- 平板倒置 37 ℃ 培养 14 h（30 ℃ 则 20 h）。菌落过多可稀释质粒后再转化；无菌落可增加至 10 μl 质粒。建议先转克隆感受态、重提质粒后再导入表达感受态。
- 挑取单菌落接种 LB 液体培养基，加入对应抗生素，220 rpm 振荡培养 14 h，按试剂盒说明提取质粒。
通用质粒转化方法
- 取100 μL感受态细胞置于冰上解冻10 min，加入2 μL质粒，冰浴30 min；随后42 ℃热激60 s，期间不要晃动，再冰浴2 min。
- 加入900 μL无抗LB液体培养基，37 ℃、180 rpm振荡培养45 min（30 ℃条件下则培养1–1.5 h）。
- 6000 rpm离心5 min，弃上清，仅保留100 μL上清重悬菌体沉淀，涂布于对应质粒抗性的LB平板上。
- 平板倒置培养，37 ℃培养14 h，或30 ℃培养20 h。
- 挑取单菌落至LB液体培养基中，加入对应抗生素，220 rpm振荡培养14 h；根据实验需求及质粒提取试剂盒说明书提取质粒。
人LARP1 ORF 克隆 (NM_033551) (编号: V029606) 注释
LOCUS V029606 10757 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V029606
VERSION V029606
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..1920
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
promoter 2010..2213
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2302..5589
/label="LARP1(NM_033551)"
/note="LARP1(NM_033551)"
/gene="LARP1"
CDS 5596..5622
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/transl_table=1
/translation="YPYDVPDYA"
/label="HA"
CDS 5626..5652
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/transl_table=1
/translation="YPYDVPDYA"
/label="HA"
CDS 5656..5682
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/transl_table=1
/translation="YPYDVPDYA"
/label="HA"
promoter 5727..5938
/note="core promoter for human elongation factor
EF-1α"
/label="EF-1α core promoter"
LTR 5951..6219
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
/label="5' LTR (truncated)"
CDS 6246..6845
/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 6852..7440
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 7514..7747
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 7819..7940
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 7959..8094
/note="SV40 origin of replication"
/label="SV40 ori"
primer_bind complement(8132..8148)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 8156..8172
/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(8180..8210)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 8225..8246
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(8534..9122)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(9293..10153)
/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(10154..10258)
/gene="bla"
/label="AmpR promoter"
primer_bind 10732..10748
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"