PCDNA3.1- EGFP

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    pCDNA3.1-EGFP

     

    PVT10754     2ug

     

    pCDNA3.1-EGFP Information

    Promoter: CMV promoter

    Replicon: pUC ori, F1 ori

    Terminator: BGH poly (A) signal

    Plasmid classification: lactation serial plasmid; lactation expression plasmid; pCDNA series plasmid.

    Plasmid size: 6173bp

    Plasmid tagging: C-EGFP

    Prokaryotic resistance: ampicillin Amp (100 ng/ml)

    Screening marker: neomycin Neo/G418

    Cloning strains: E. coli DH5 and E.

    Culture conditions: 37 C, aerobic, LB

    Expression host: mammalian cells such as 293T

    Induction mode: no need to induce

    5'sequencing primers: pCDNA3.1-F (CTAGAGAACCCACTGCTTAC)

    3'sequencing primers: pCDNA3.1-R (TAGAAGGCACAGTCGAGG)

    Note: Restriction site is different from PVT10755 pCDNA3.1-EGFP-2

     

    pCDNA3.1-EGFP Description

    pCDNA3.1-EGFP is a Mammalian fluorescent plasmid.

    pCDNA3.1-EGFP  was obtained by cloning the EGFP gene into the pCDNA3.1 vector. It was designed for stable and transient expression in mammalian hosts. Most mammalian cells can perform high levels of stable and non replicating transient expression. Human cytomegalovirus immediate early (CMV) promoter is used for high level expression in a wide range of mammalian cells. Abnormal replication in cell lines with latent infection of SV40 or expression of SV40 large T antigen (e.g. COS-1, COS-7).

    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.

     

    pCDNA3.1-EGFP Multiple cloning site

     

    pCDNA3.1-EGFP Sequence

    LOCUS       Exported                6173 bp ds-DNA     circular SYN 07-12-2015
    DEFINITION  synthetic circular DNA
    KEYWORDS    pCDNA3.1(+)-EGFP
    SOURCE      synthetic DNA construct
      ORGANISM  synthetic DNA construct
    REFERENCE   1  (bases 1 to 6173)
      TITLE     Direct Submission
      JOURNAL   Exported 2016-9-18
    FEATURES             Location/Qualifiers
         source          1..6173
                         /organism="synthetic DNA construct"
                         /mol_type="other DNA"
         enhancer        236..615
                         /note="CMV enhancer"
                         /note="human cytomegalovirus immediate early enhancer"
         promoter        616..819
                         /note="CMV promoter"
                         /note="human cytomegalovirus (CMV) immediate early
                         promoter"
         promoter        864..882
                         /note="T7 promoter"
                         /note="promoter for bacteriophage T7 RNA polymerase"
         CDS             1003..1722
                         /codon_start=1
                         /product="enhanced GFP"
                         /note="EGFP"
                         /note="mammalian codon-optimized"
                         /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                         KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                         GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                         VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                         EFVTAAGITLGMDELYK"
         polyA_signal    1774..1998
                         /note="bGH poly(A) signal"
                         /note="bovine growth hormone polyadenylation signal"
         rep_origin      2044..2472
                         /direction=RIGHT
                         /note="f1 ori"
                         /note="f1 bacteriophage origin of replication; arrow
                         indicates direction of (+) strand synthesis"
         promoter        2486..2815
                         /note="SV40 promoter"
                         /note="SV40 enhancer and early promoter"
         rep_origin      2666..2801
                         /note="SV40 ori"
                         /note="SV40 origin of replication"
         CDS             2882..3676
                         /codon_start=1
                         /gene="aph(3')-II (or nptII)"
                         /product="aminoglycoside phosphotransferase from Tn5"
                         /note="NeoR/KanR"
                         /note="confers resistance to neomycin, kanamycin, and G418
                         (Geneticin(R))"
                         /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                         VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                         SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                         GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                         LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
         polyA_signal    3850..3971
                         /note="SV40 poly(A) signal"
                         /note="SV40 polyadenylation signal"
         primer_bind     complement(4020..4036)
                         /note="M13 rev"
                         /note="common sequencing primer, one of multiple similar
                         variants"
         protein_bind    4044..4060
                         /bound_moiety="lac repressor encoded by lacI"
                         /note="lac operator"
                         /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-beta-D-thiogalactopyranoside (IPTG)."
         promoter        complement(4068..4098)
                         /note="lac promoter"
                         /note="promoter for the E. coli lac operon"
         protein_bind    4113..4134
                         /bound_moiety="E. coli catabolite activator protein"
                         /note="CAP binding site"
                         /note="CAP binding activates transcription in the presence
                         of cAMP."
         rep_origin      complement(4422..5007)
                         /direction=LEFT
                         /note="ori"
                         /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                         replication"
         CDS             complement(5178..6038)
                         /codon_start=1
                         /gene="bla"
                         /product="beta-lactamase"
                         /note="AmpR"
                         /note="confers resistance to ampicillin, carbenicillin, and
                         related antibiotics"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                         ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                         PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                         EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                         LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                         LIKHW"
         promoter        complement(6039..6143)
                         /gene="bla"
                         /note="AmpR promoter"
    ORIGIN
            1 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
           61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
          121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
          181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
          241 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
          301 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
          361 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
          421 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
          481 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
          541 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
          601 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
          661 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
          721 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
          781 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
          841 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agctggctag
          901 cgtttaaact taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
          961 cagtcgacgg taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag
         1021 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
         1081 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
         1141 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
         1201 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
         1261 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
         1321 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
         1381 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
         1441 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
         1501 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
         1561 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
         1621 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
         1681 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg ctcgagtcta
         1741 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
         1801 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
         1861 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
         1921 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
         1981 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
         2041 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
         2101 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
         2161 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
         2221 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
         2281 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
         2341 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
         2401 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
         2461 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
         2521 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
         2581 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
         2641 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
         2701 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
         2761 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
         2821 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
         2881 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
         2941 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
         3001 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
         3061 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
         3121 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
         3181 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
         3241 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
         3301 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
         3361 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
         3421 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
         3481 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
         3541 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
         3601 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
         3661 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
         3721 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
         3781 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
         3841 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
         3901 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
         3961 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
         4021 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
         4081 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
         4141 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
         4201 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
         4261 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
         4321 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
         4381 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
         4441 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
         4501 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
         4561 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
         4621 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
         4681 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
         4741 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
         4801 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
         4861 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
         4921 agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag
         4981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
         5041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
         5101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
         5161 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
         5221 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
         5281 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
         5341 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
         5401 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
         5461 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
         5521 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
         5581 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
         5641 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
         5701 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
         5761 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
         5821 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
         5881 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
         5941 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
         6001 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
         6061 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
         6121 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgt
    //

     

    Caution:
    1.  This product is FOR RESEARCH USE ONLY!
    2.  The item is lyophilized form, Please take the powder plasmid by centrifugation at 5000rpm/min for 1min. Add 20μl ddH2O in to the tube of plasmid.