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网站首页 >> pCDNA3.1-EGFP①哺乳荧光质粒 货号：P2087
pCDNA3.1-EGFP①哺乳荧光质粒 货号：P2087

质粒简介

        pCDNA3.1-EGFP质粒是将EGFP基因克隆进入pCDNA3.1载体所得，设计用于哺乳动物宿主中的高水平稳定和瞬时表达。大多数哺乳动物细胞可以进行高水平的稳定和非复制性瞬时表达。人类巨细胞病毒立即早期（CMV）启动子，用于在广泛哺乳动物细胞中的高水平表达。在潜伏感染SV40或表达SV40大T抗原（例如COS-1，COS-7）的细胞系中的异常复制。
      The GFP in pcDNA3.1-eGFP stands for green fluorescent protein. Deoxyribonucleic acids (DNAs) are typically made up of protein, so it makes sense for a special protein to make up a specific type of DNA add-gene such as the pcDNA3.1.although other means besides cloning would also be openly discussed. In this study, a detailed overview of how pcDNA-eGFP can be produced through cloning is done thanks to the use of secondary sources; a proper review of the pcDNA3.1-eGFP is first carried out to understand this compound. Afterward, the importance of a vector in the cloning process is looked at in detail and finally the cloning system is covered extensivel GFP proteins are generally composed of 238 amino acids with molecular masses of around 26.9 KD [1] and are typically used in the field of molecular biology as one of the many reporter genes. Basically, a reporter gene is a type of gene that researchers, especially in laboratory experiments, use to attach to a pre-specified sequence of the gene (oftentimes an experimental one) such as that of bacteria, plants, animals, and cell cultures. Some of the criteria that researchers use when selecting a reporter gene include, but may not be limited to, having easily identifiable and selectable markers and their ability to introduce changes that tend to be easily spotted under certain conditions. When conducting laboratory experiments in molecular biology, it would be important for researchers to know the different variables involved and the impact that they create on the experimental environment. Therefore, it makes sense to select reporter genes that possess these qualities such as the GFP [1]. In previously published studies involving the use of GFPs, including but not limited to pcDNA3.1, it has been common for researchers to introduce the GFP gene into cells using vector-based systems. In some cases, the researchers also used recombinant viruses (attaching the GFP to them). Being used as a reporter protein, the location of the target protein can be easily identified and expressed. However, in many of the laboratory experiments, the selection market of the GFPs used was not specific enough and there is often no selection market to normalize the transfection among other reactions.

  1. 此载体骨架为Invitrogen公司的pCDNA3.1(+)。

  2. EGFP插入位点为EcoRI、NotI。

  3. 此载体已验证：将此载体转染至293细胞、Hela细胞、CHO细胞等多株细胞，24小时后观察荧光，可见多且亮的绿色荧光。

  4. 此载体可作为pCDNA3.1(+)系列的NC对照载体使用，亦可用来构建【目的基因-EGFP基因】融合形式的基因过表达载体。

  5. 此载体带有Neo抗性基因，可在真核细胞中用G418进行筛选。

Location of features

232-819：CMV promoter

995-1718：EGFP

1773-1997：BGH polyA

2043-2471：f1 ori

2476-2819：SV40 promoter

2881-3675：Neo(R)

3849-3979：SV40 polyA

4362-5032：pUC ori（反向）

5177-6037：Amp(R)（反向）

6038-6136：bla promoter（反向）

Primer

CMV-f（通用引物）：769-789

Sequence：5’-CGCAAATGGGCGGTAGGCGTG-3’

BGH-r（通用引物）：1784-1767

Sequence：5’-TAGAAGGCACAGTCGAGG-3’

Propagation in E.coli

  1. DH5α、JM101、XL1-Blue等均可。

  2. 抗性为Amp+。

  3. Copy number：高（~500）


    质粒信息

别名 和2酶切位点不一样
启动子 CMV
复制子 pUC
终止子 BGH poly（A）signal
质粒分类 哺乳系列质粒；哺乳表达质粒；pCDNA系列质粒
质粒大小 6173bp
质粒标签 C-EGFP
原核抗性 Amp
真核抗性 G418
克隆菌株 DH5α
培养条件 37℃
表达宿主 293T等哺乳细胞
培养条件 37℃，5%CO2
诱导方式 无需诱导
5'测序引物 pCDNA3.1-F（CTAGAGAACCCACTGCTTAC）
3'测序引物 pCDNA3.1-R（TAGAAGGCACAGTCGAGG）
质粒宿主 哺乳细胞
质粒用途 蛋白表达
片段类型 基因CDS
原核抗性 氨苄青霉素

图谱




序列 
ID   pcDNA3.1-EGFP standard; circular DNA; SYN; 6173 BP.
DT   28-AUG-2003
CC   This file is created by Vector NTI
CC   http://www.yrgene.com/
CC   ORIGDB|GenBank
CC   VNTDATE|626698292|
CC   VNTDBDATE|-12278268|
CC   LSOWNER|
CC   VNTAUTHORNAME|YRgene|
FH   Key             Location/Qualifiers
FH
FT   CDS             complement(5177..6037)
FT                   /invitrogen="1020000"
FT                   /vntifkey="4"
FT                   /label=Amp+
FT   polyA_site      1773..1997
FT                   /invitrogen="1870000"
FT                   /vntifkey="26"
FT                   /label=BGHpolyA
FT   promoter        232..819
FT                   /invitrogen="2180000"
FT                   /vntifkey="29"
FT                   /label=CMVpromoter
FT   rep_origin      2043..2471
FT                   /invitrogen="2500000"
FT                   /vntifkey="33"
FT                   /label=f1origin
FT   CDS             2881..3675
FT                   /invitrogen="1260000"
FT                   /vntifkey="4"
FT                   /label=Neo+
FT   rep_origin      complement(4362..5032)
FT                   /invitrogen="2540000"
FT                   /vntifkey="33"
FT                   /label=pUCorigin
FT   promoter        2476..2819
FT                   /invitrogen="2290000"
FT                   /vntifkey="29"
FT                   /label=SV40earlypromoter
FT   polyA_signal    3849..3979
FT                   /invitrogen="1840000"
FT                   /vntifkey="25"
FT                   /label=SV40pA
FT   CDS             1002..1721
FT                   /vntifkey="4"
FT                   /label=EGFP
SQ   Sequence 6173 BP; 1426 A; 1664 C; 1599 G; 1484 t;
     gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg        60
     ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg       120
     cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc       180
     ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt       240
     gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata       300
     tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc       360
     cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc       420
     attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt       480
     atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt       540
     atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca       600
     tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg       660
     actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc       720
     aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg       780
     gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca       840
     ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc       900
     gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc       960
     agtcgacggt accgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg      1020
     agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca      1080
     agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt      1140
     tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct      1200
     acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt      1260
     ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact      1320
     acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga      1380
     agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca      1440
     acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca      1500
     agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca      1560
     cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg      1620
     ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg      1680
     ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc tcgagtctag      1740
     agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt      1800
     tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc      1860
     ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg      1920
     tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga      1980
     tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc      2040
     ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac      2100
     cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc      2160
     cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt      2220
     tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg      2280
     gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag      2340
     tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt      2400
     ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt      2460
     taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc      2520
     ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag      2580
     tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc      2640
     atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct      2700
     ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct      2760
     gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc      2820
     ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc      2880
     atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc      2940
     ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca      3000
     gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg      3060
     caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg      3120
     ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag      3180
     gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg      3240
     cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc      3300
     atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa      3360
     gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac      3420
     ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat      3480
     ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac      3540
     atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc      3600
     ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt      3660
     gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc      3720
     tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg      3780
     ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg      3840
     cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa      3900
     atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca      3960
     atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt      4020
     catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg      4080
     gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt      4140
     tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg      4200
     gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg      4260
     actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa      4320
     tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc      4380
     aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc      4440
     ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat      4500
     aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc      4560
     cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct      4620
     cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg      4680
     aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc      4740
     cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga      4800
     ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa      4860
     gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta      4920
     gctcttgatc cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga      4980
     ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg      5040
     ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct      5100
     tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt      5160
     aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc      5220
     tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg      5280
     gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag      5340
     atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt      5400
     tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag      5460
     ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt      5520
     ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca      5580
     tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg      5640
     ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat      5700
     ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta      5760
     tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca      5820
     gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct      5880
     taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat      5940
     cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa      6000
     agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt      6060
     gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa      6120
     ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc             6173
//

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