pCDH-ECMV-Neu1(mouse)-3xFLAG-EF1α-ZsGreen1-Puro9744 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800920096005' LTR (truncated)HIV-1 ΨRREgp41 peptidecPPT/CTSCMV enhancerCMV promoterNeu1(NM_010893)3xFLAGEF-1α core promoter5' LTR (truncated)ZsGreen1T2APuroRWPRE3' LTR (ΔU3)SV40 poly(A) signalSV40 oriM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterM13 fwd

基本信息

载体名称
pCDH-ECMV-Neu1(mouse)-3xFLAG-EF1α-ZsGreen1-Puro
载体抗性
Ampicillin
Refseq ID
NM_010893.3
载体长度
9744 bp
载体类型
Protein expression
复制子
ori
宿主
Mammalian cells, Lentivirus
筛选标记
Puro;ZsGreen1
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
Mus musculus neuraminidase 1 (Neu1), mRNA

质粒操作方法

质粒复溶方法

  1. 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。
  2. 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。
  3. 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。
  4. 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)。
  5. 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。
  6. 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。
  7. 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

  1. 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。
  2. 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)。
  3. 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。
  4. 平板倒置培养,37 ℃培养14 h,或30 ℃培养20 h。
  5. 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。

小鼠Neu1 ORF 克隆 (NM_010893) (编号: V027107) 注释

LOCUS       V027107                 9744 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V027107
VERSION     V027107
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             1494..1538
                     /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    1801..1916
                     /note="central polypurine tract and central termination
                     sequence of HIV-1 (lacking the first T)"
                     /label="cPPT/CTS"
     enhancer        1974..2277
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2278..2481
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2588..3814
                     /label="Neu1(NM_010893)"
                     /note="Neu1(NM_010893)"
                     /gene="Neu1"
     CDS             3839..3910
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags"
                     /transl_table=1
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
                     /label="3xFLAG"
     promoter        3964..4175
                     /note="core promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             4188..4456
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     CDS             4484..5176
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="Zoanthus green fluorescent protein"
                     /transl_table=1
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
                     /label="ZsGreen1"
     CDS             5186..5239
                     /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             5240..5839
                     /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    5840..6427
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6501..6734
                     /note="self-inactivating 3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    6806..6927
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6946..7081
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(7119..7135)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7143..7159
                     /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(7167..7197)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7212..7233
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7521..8109)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8280..9140)
                     /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(9141..9245)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     9719..9735
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"