pEGFP- C1 Plasmid

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pEGFP- C1 Plasmid

PVT1205    2ug
 

pEGFP-C1 Information

Promoter: CMV promoter

Replicon: F1 ori, pUC ori, SV40 ori

Terminator: SV40 poly (A) signal

Plasmid Classification: Mammalian Series Plasmids; Mammalian Fluorescent Plasmids; Mammalian Green Plasmids

Plasmid size: 4731 BP

Plasmid label: C-EGFP

Prokaryotic resistance: Kanamycin Kan (50 ug/ml)

Screening marker: neomycin Neo/G418

Cloning strains: Escherichia coli such as DH5alpha

Culture conditions: 37 C, aerobic LB

Expressing host: mammalian cells such as 293T

Culture conditions: 37 C, 5% CO2

Induction mode: no induction, transient expression

5'Sequencing Primers: pEGFP-C-5

3'Sequencing Primers: pEGFP-C-3

 

pEGFP-C1 Description

PEGFP-C1 encodes a red-shifted variant of wild-type GFP, which has been optimized for brighter fluorescence and higher expression in mammalian cells. (maximum excitation = 488 nm; maximum emission = 507 nm) pEGFP-C1 encodes GFPmut1 variant, which contains Phe-64 pairs of Leu and Ser-65 to Thr double amino acids instead of MCS in pEGFPC1. If the gene cloned into MCS between EGFP coding sequence and SV40 polyA is in the same reading frame as that of EGFP and there is no intermediate termination codon, it is expressed as a C-terminal fusion of EGFP. The SV40 polyadenylate signal downstream of EGFP gene directly and appropriately processes the 3'terminal of EGFP gene. The carrier skeleton also contains the source of SV40 for replication in mammalian cells expressing SV40 T antigen. Neor, composed of the early promoter of SV40, the neomycin/kanamycin resistance gene of Tn5 and the polyadenylation signal from the herpes simplex virus thymidine kinase (HSV TK) gene, allows the selection of stable transfected eukaryotic cells using G418. The bacterial promoter upstream of the box expressed kanamycin resistance in E. coli. PEGFP-C1backbone also provides a starting point for pUC replication for reproduction in E. coli and a source of F1 for single-stranded DNA production.

 pEGFP-C1encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm;emission maximum = 507 nm.) pEGFP-C1 encodes the GFPmut1 variant (4) 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 (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFPC1is 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-C1backbone 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-C1 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-C1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
Propagation in E. coli

        • 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.
        • E. coli replication origin: pUC
        • Copy number: ≈500
        • Plasmid incompatibility group: pMB1/ColE1

 

pEGFP-C1 Multiple cloning site

pEGFP-C1-vector

 

pEGFP-C1 Sequence

LOCUS       Exported                4731 bp ds-DNA     circular SYN 30-AUG-2016

DEFINITION  synthetic circular DNA

KEYWORDS    Untitled 8

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4731)

  TITLE     Direct Submission

  JOURNAL   Exported Tuesday, August 30, 2016

FEATURES             Location/Qualifiers

     source          1..4731

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /note="CMV enhancer"

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        365..568

                     /note="CMV promoter"

                     /note="human cytomegalovirus (CMV) immediate early 

                     promoter"

     CDS             613..1329

                     /codon_start=1

                     /product="enhanced GFP"

                     /note="EGFP"

                     /note="mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     misc_feature    1330..1395

                     /note="MCS"

                     /note="multiple cloning site"

     polyA_signal    1519..1640

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1647..2102)

                     /direction=LEFT

                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        2129..2233

                     /gene="bla"

                     /note="AmpR promoter"

     promoter        2235..2592

                     /note="SV40 promoter"

                     /note="SV40 enhancer and early promoter"

     rep_origin      2443..2578

                     /note="SV40 ori"

                     /note="SV40 origin of replication"

     CDS             2627..3421

                     /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

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3653..3700

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

                     /note="herpes simplex virus thymidine kinase 

                     polyadenylation signal (Cole and Stacy, 1985)"

     rep_origin      4029..4617

                     /direction=RIGHT

                     /note="ori"

                     /note="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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc

     1381 gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta

     1441 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg

     1501 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat

     1561 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc

     1621 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc

     1681 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc

     1741 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag

     1801 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga

     1861 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc

     1921 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa

     1981 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt

     2041 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc

     2101 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat

     2161 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg

     2221 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt

     2281 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca

     2341 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat

     2401 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg

     2461 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc

     2521 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta

     2581 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga

     2641 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc

     2701 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc

     2761 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc

     2821 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac

     2881 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc

     2941 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac

     3001 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg

     3061 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct

     3121 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt

     3181 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg

     3241 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac

     3301 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg

     3361 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg

     3421 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat

     3481 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc

     3541 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg

     3601 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata

     3661 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc

     3721 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt

     3781 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac

     3841 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta

     3901 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc

     3961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa

     4021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca

     4081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta

     4141 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc

     4201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca

     4261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta

     4321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag

     4381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt

     4441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc

     4501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac

     4561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac

     4621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc

     4681 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t

//


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Product is for research use only!

 

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