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pCMV-N-Flag
产品编号: D2722-1μg
产品包装: 1μg
产品价格: 888.00元
产品简介
使用说明
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产品编号 产品名称 产品包装 产品价格
D2722-1μg pCMV-N-Flag 1μg 888.00元

      pCMV-N-Flag是碧云天自行研发的用于在哺乳动物细胞中表达N端和Flag tag(Flag标签)融合的目的蛋白的表达质粒。含有CMV启动子可以高效启动目的蛋白在细胞中的表达;在多克隆位点的5'端含有一个可以编码Flag标签的序列,因此可以表达出含有Flag标签的融合蛋白,可以方便地使用抗Flag的抗体来识别目的蛋白,有利于目的蛋白检测和分离纯化。质粒为卡那霉素抗性。转染细胞后,可使用G418筛选稳定表达目的蛋白的细胞株。
      pCMV-N-Flag质粒的主要信息如下:

Feature Nucleotide Position
CMV promoter 1-602
T3 promoter and T3 primer binding site 620-639
Flag tag 679-702
multiple cloning site 705-778
T7 promoter and T7 primer binding site 821-842
SV40 polyA signal 854-1237
f1 origin of ss-DNA replication 1375-1681
bla promoter 1706-1830
SV40 promoter 1850-2188
neomycin/kanamycin resistance ORF 2223-3014
HSV-thymidine kinase(TK)polyA signal 3015-3473
pUC origin 3602-4269

      pCMV-N-Flag质粒的图谱如下:

      pCMV-N-Flag的多克隆位点的详细图谱如下:

Flag tag
SacI M D Y K D D D D K
651 GAGCTCCACC GCGGTGGCGG
CCGCC
ATG GA
TTACAAGGAT
GACGACGATA
CTCGAGGTGG CGCCACCGCC
GGCGG
TAC CT
AATGTTCCTA
CTGCTGCTAT
XmaI PstI
SmaI BamHI HindIII EcoRI EcoRV SalI BglII
701
AG AGCCCGGG
CGGATCCAAG CTTCTGCAGG AATTCGATAT CGTCGACAGA
TC TCGGGCCC
GCCTAGGTTC GAAGACGTCC TTAAGCTATA GCAGCTGTCT
XhoI XbaI ApaI
751 TCTCTCGAGT CTAGAACTAG TGGGCCCGGT ACCTTAATTA ATTAAGGTAC
AGAGAGCTCA GATCTTGATC ACCCGGGCCA TGGAATTAAT TAATTCCATG

      pCMV-N-Flag中没有的酶切位点(Restriction enzymes that do not cut pCMV-N-Flag)包括:

Afl II Age I Ahd I Asc I Bbs I Bbv II Blp I
Bsg I BsiW I BsmB I BspM II BsrG I BssH II Bst1107 I
BstE II Ear I Eco47 III Eco72 I EcoN I Esp I Fse I
Nru I PflM I Pme I Pml I PpuM I Psp1406 I Sap I
Sca I Spl I

      pCMV-N-Flag中的单酶切位点(Restriction enzymes that cut pCMV-N-Flag once)包括:

Nde I CA`TA,TG 241 Pvu I CG,AT`CG 855
SnaB I TAC|GTA 347 Bcl I T`GATC,A 1009
Nhe I G`CTAG,C 598 Mun I C`AATT,G 1102
Sac I G,AGCT`C 656 Hpa I GTT|AAC 1115
Sac II CC,GC`GG 663 Mlu I A`CGCG,T 1238
BstX I CCAN,NNNN`NTGG 664 Dra III CAC,NNN`GTG 1468
Not I GC`GGCC,GC 669 Sfi I GGCCN,NNN`NGGCC 2127
PspA I C`CCGG,G 706 BseR I GAGGAG 16/14 2170
Xma I C`CCGG,G 706 Stu I AGG|CCT 2173
Srf I GCCC|GGGC 708 Cla I AT`CG,AT 2192
Sma I CCC|GGG 708 Kas I G`GCGC,C 2351
BamH I G`GATC,C 713 Nar I GG`CG,CC 2352
Hind III A`AGCT,T 719 Ehe I GGC|GCC 2353
Pst I C,TGCA`G 729 Bbe I G,GCGC`C 2355
EcoR I G`AATT,C 731 Msc I TGG|CCA 2434
EcoR V GAT|ATC 739 Tth111 I GACN`N,NGTC 2470
Sal I G`TCGA,C 743 BsrD I GCAATG, 8 2585
Acc I GT`MK,AC 744 Bsp1286 I G,DGCH`C 2655
Bgl II A`GATC,T 749 Rsr II CG`GWC,CG 2868
PaeR7 I C`TCGA,G 755 BsiC I TT`CG,AA 3034
Xho I C`TCGA,G 755 BstB I TT`CG,AA 3034
Xba I T`CTAG,A 761 Bsa I GGTCTC 7/11 3341
Spe I A`CTAG,T 767 HgiE II ACCNNNNNNGGT-1/13 3681
Bsp120 I G`GGCC,C 773 ApaL I G`TGCA,C 3956
Apa I G,GGCC`C 777

      pCMV-N-Flag质粒中对于插入片段进行测序时,推荐使用的正向测序引物T3和反向测序引物T7的序列如下:
      T3 primer(620-639): 5′AATTAACCCTCACTAAAGGG 3′
      T7 primer(821-842): 5′GTAATACGACTCACTATAGGGC 3′
      pCMV-N-Flag的全序列信息:D2722 pCMV-N-Flag-seq.txt
包装清单:

产品编号 产品名称 包装
D2722-1μg pCMV-N-Flag 1μg
D2722-001μg pCMV-N-Flag 100μg
说明书 1份

保存条件:
      -20℃保存。
注意事项
      本质粒未经碧云天书面许可不得用于任何商业用途,也不得移交给订货人所在实验室外的任何个人或单位。
      本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
      为了您的安全和健康,请穿实验服并戴一次性手套操作。

使用说明:
1. 首次使用时请先取少量本质粒转化大肠杆菌,进行质粒小量、中量或大量抽提后再用于后续用途。抽提获得的质粒可以通过酶切电泳进行鉴定,或通过测序进行鉴定。
2. 100μg包装的本产品质粒浓度为0.1μg/μl,共1ml。可以直接用于酶切或者转染细胞。
3. pCMV-N-Flag质粒在其多克隆位点适当酶切后可以插入待表达的目的基因,需注意插入基因片段和tag之间的读码框要一致,即需要避免发生移码突变。构建的质粒可以用常规方法转染细胞。

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