pEGFP- C2 Plasmid

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  • Model: PVT1206
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pEGFP-C2 Plasmid

PVT1206      2ug
 

pEGFP- C2 Plasmid Information

Promoter: CMV promoter

Replicator: pUC ori, F1 ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation serial plasmids; lactating fluorescent plasmid; lactation green plasmid

Plasmid size: 4735bp

Plasmid label: C-EGFP

Prokaryotic resistance: kanamycin Kan (50 mu g/ml)

Screening markers: neomycin Neo/G418

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 C, 5%CO2

Induction mode: no induction, instantaneous expression

5'sequencing primers: pEGFP-C-5' (CATGGTCCTGCTGGAGTTCGTG)

3'sequencing primers: pEGFP-C-3' (TATGGCTGATTATGATCAGT)

 

pEGFP- C2 Plasmid Description

pEGFP-C2 Plasmid encodes a red-shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-C2 encodes the GFPmut1 variant which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences . Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-C2 is between the EGFP coding sequences and the SV40 poly A. Genes cloned into the MCS will be expressed as fusions to the C terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-C2 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. Fusions to the C terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo. The target gene should be cloned into pEGFP-C2 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418. pEGFP-C2 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker). Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue. Selectable marker: plasmid confers resistance to kanamycin (30 µg/ml) to E. coli hosts.

 

pEGFP- C2 Plasmid multiple site

pEGFP-C2-site

 

pEGFP- C2  Plasmid Sequence

LOCUS       Exported                4735 bp ds-DNA     circular SYN 31-AUG-2016

DEFINITION  synthetic circular DNA

KEYWORDS    pEGFP-C2

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4735)

  TITLE     Direct Submission

  JOURNAL   Exported 2015-5-31

FEATURES             Location/Qualifiers

     source          1..4735

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /note="CMV enhancer"

                     /note="CMV enhancer;human cytomegalovirus immediate early 

                     enhancer"

     promoter        365..568

                     /note="CMV promoter"

                     /note="CMV promoter;human cytomegalovirus (CMV) immediate 

                     early promoter"

     CDS             613..1329

                     /codon_start=1

                     /product="enhanced GFP"

                     /note="enhanced GFP"

                     /note="EGFP;mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     misc_feature    1343..1399

                     /note="MCS"

                     /note="MCS;multiple cloning site"

     polyA_signal    1523..1644

                     /note="SV40 poly(A) signal"

                     /note="SV40 poly(A) signal;SV40 polyadenylation signal"

     rep_origin      complement(1651..2106)

                     /direction=LEFT

                     /note="f1 ori"

                     /note="f1 ori;f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        2133..2237

                     /gene="bla"

                     /note="bla AmpR promoter"

                     /note="AmpR promoter"

     promoter        2239..2596

                     /note="SV40 promoter"

                     /note="SV40 promoter;SV40 enhancer and early promoter"

     rep_origin      2447..2582

                     /note="SV40 ori"

                     /note="SV40 ori;SV40 origin of replication"

     CDS             2631..3425

                     /codon_start=1

                     /gene="aph(3')-II (or nptII)"

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /note="aph(3')-II (or nptII)"

                     /note="NeoR/KanR;confers resistance to neomycin, kanamycin,

                     and G418 (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3657..3704

                     /note="HSV TK poly(A) signal"

                     /note="HSV TK poly(A) signal;herpesvirus thymidine kinase 

                     polyadenylation signal"

     rep_origin      4033..4621

                     /direction=RIGHT

                     /note="ori"

                     /note="ori;high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

ORIGIN

        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg

       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt

      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca

      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc

      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta

      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac

      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg

      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg

      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt

      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta

      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg

      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc

      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg

      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc

      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag

      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag

      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac

     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac

     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc

     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg

     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc

     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag

     1321 ctgtacaagt ccggccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg

     1381 taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt

     1441 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa

     1501 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag

     1561 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt

     1621 gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat

     1681 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa

     1741 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca

     1801 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg

     1861 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta

     1921 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg

     1981 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa

     2041 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg

     2101 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct

     2161 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat

     2221 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg

     2281 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta

     2341 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat

     2401 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac

     2461 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga

     2521 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg

     2581 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac

     2641 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact

     2701 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc

     2761 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg

     2821 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg

     2881 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt

     2941 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc

     3001 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag

     3061 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg

     3121 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc

     3181 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt

     3241 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg

     3301 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt

     3361 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct

     3421 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg

     3481 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga

     3541 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag

     3601 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc

     3661 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg

     3721 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg

     3781 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc

     3841 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga

     3901 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat

     3961 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat

     4021 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa

     4081 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa

     4141 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt

     4201 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt

     4261 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata

     4321 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt

     4381 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac

     4441 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga

     4501 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg

     4561 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa

     4621 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat

     4681 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat

//

 

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Product is for research use only!

 

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pEGFP-C2,Plasmid pEGFP-C2,pEGFP-C2 vector

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