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PVT1207   2ug

pEGFP-C3 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: 4727bp

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-C3 Description

PEGFP-C3 has optimized the red shift variant of the encoded wild type GFP to have a brighter fluorescence, high expression in mammalian cells (maximum excitation wavelength = 488nm, maximum release wavelength = 507nm). The coding sequence of EGFP gene contains more than 190 silent base changes, which conforms to human codon usage preference. One side of the EGFP sequence is KozaK, which further improves the efficiency of translation in eukaryotic cells.

  pEGFP-C3 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-C3 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 (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-C3 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-C3 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-C3 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-C3 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.



pEGFP-C3 Sequence

LOCUS       Exported                4727 bp ds-DNA    circular SYN 18-10-2015

KEYWORDS    Untitled 2

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4727)

  AUTHORS   admin

  TITLE     Direct Submission

  JOURNAL   Exported 2015-10-18

FEATURES             Location/Qualifiers

     source          1..4727

                     /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 


     CDS             613..1329


                     /product="enhanced GFP"


                     /note="mammalian codon-optimized"






     misc_feature    1335..1391


                     /note="multiple cloning site"

     polyA_signal    1515..1636

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1643..2098)


                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        2125..2229


                     /note="AmpR promoter"

     promoter        2231..2588

                     /note="SV40 promoter"

                     /note="SV40 enhancer and early promoter"

     rep_origin      2439..2574

                     /note="SV40 ori"

                     /note="SV40 origin of replication"

     CDS             2623..3417


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

                     /product="aminoglycoside phosphotransferase from Tn5"


                     /note="confers resistance to neomycin, kanamycin, and G418 







     polyA_signal    3649..3696

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

                     /note="herpesvirus thymidine kinase polyadenylation signal"

     rep_origin      4025..4613



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



        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 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg

     1381 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg

     1441 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg

     1501 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca

     1561 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac

     1621 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta

     1681 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat

     1741 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca

     1801 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc

     1861 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta

     1921 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg

     1981 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg

     2041 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca

     2101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat

     2161 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa

     2221 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa

     2281 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa

     2341 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca

     2401 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca

     2461 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg

     2521 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct

     2581 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga

     2641 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa

     2701 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt

     2761 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg

     2821 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa

     2881 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac

     2941 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt

     3001 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact

     3061 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg

     3121 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg

     3181 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc

     3241 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt

     3301 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc

     3361 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg

     3421 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg

     3481 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct

     3541 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc

     3601 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa

     3661 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg

     3721 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt

     3781 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg

     3841 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac

     3901 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa

     3961 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat

     4021 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc

     4081 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg

     4141 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc

     4201 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg

     4261 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg

     4321 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa

     4381 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg

     4441 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga

     4501 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct

     4561 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca

     4621 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc

     4681 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat



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pEGFP-C3,Plasmid pEGFP-C3,pEGFP-C3 vector

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