pEGFP- N3

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  • Model: PVT1204
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pEGFP-N3

PVT1204 2ug

 

pEGFP-N3 Information

Promoter: CMV

Replicon: pUC ori

Terminator: SV40 poly (A) signal

Plasmid size: 4729 BP

Prokaryotic resistance: kanamycin Kan

Screening Marker: Hereditary Mycin G418

Cloning strain: Escherichia coli DH5alpha

Culture conditions: 37 C, aerobic, LB

Expression host: mammalian cells

Induction mode: no induction, transient expression

5'Sequencing Primers: CMV-F: CGCAAATGGGGGGGTAGGGGGGTG

3'Sequencing Primers: Designing Primers Based on Sequences

 

pEGFP-N3 Description

pEGFP-N3 encodes 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-N3 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 pEGFP-N3 is between the immediate early promoter of CMV (PCMV IE) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N 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-N3 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

 

pEGFP-N3 Sites

pEGFP-N3-vector

 

pEGFP-N3 Sequence

LOCUS       Exported                4729 bp ds-DNA    circular SYN 18-10-2015

FEATURES             Location/Qualifiers

     source          1..4729

                     /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"

     misc_feature    609..665

                     /note="MCS"

                     /note="multiple cloning site"

     CDS             675..1394

                     /codon_start=1

                     /product="enhanced GFP"

                     /note="EGFP"

                     /note="mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     polyA_signal    1517..1638

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1645..2100)

                     /direction=LEFT

                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        2127..2231

                     /gene="bla"

                     /note="AmpR promoter"

     promoter        2233..2590

                     /note="SV40 promoter"

                     /note="SV40 enhancer and early promoter"

     rep_origin      2441..2576

                     /note="SV40 ori"

                     /note="SV40 origin of replication"

     CDS             2625..3419

                     /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    3651..3698

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

                     /note="herpesvirus thymidine kinase polyadenylation signal"

     rep_origin      4027..4615

                     /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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccatcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc

      721 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg

      781 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg

      841 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc

      901 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg

      961 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg

     1021 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca

     1081 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg

     1141 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca

     1201 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc

     1261 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc

     1321 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg

     1381 agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga

     1441 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa

     1501 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag

     1561 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa

     1621 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt

     1681 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt

     1741 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc

     1801 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg

     1861 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac

     1921 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg

     1981 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag

     2041 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt

     2101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac

     2161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa

     2221 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg

     2281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc

     2341 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct

     2401 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc

     2461 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga

     2521 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg

     2581 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg

     2641 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac

     2701 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg

     2761 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc

     2821 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg

     2881 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc

     2941 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc

     3001 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta

     3061 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg

     3121 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg

     3181 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat

     3241 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc

     3301 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta

     3361 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag

     3421 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt

     3481 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg

     3541 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag

     3601 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa

     3661 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca

     3721 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc

     3781 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt

     3841 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa

     3901 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa

     3961 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg

     4021 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc

     4081 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac

     4141 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca

     4201 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt

     4261 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc

     4321 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg

     4381 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc

     4441 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac

     4501 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct

     4561 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc

     4621 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt

     4681 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat

//

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pEGFP-N3,Plasmid pEGFP-N3,pEGFP-N3 vector

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