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基本信息

質粒圖譜和多克隆位點信息

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載體描述   

pEGFP-N1編碼野生型GFP的一個紅移變體，已被優化，有更亮的熒光，在哺乳細胞有更高的表達，（最大激發波長=488nm，最大釋放波長=507nm）。EGFP基因編碼序列，含有超過190個沉默堿基的變化，這符合人類密碼子使用偏好。

EGFP序列的一側已經轉變成和KozaK一致的翻譯起始位點，進一步提高了在真核細胞的翻譯效率。

pEGFP-N1 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-N1 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-N1 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-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應用

Fusions to the N 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-N1 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).

載體序列

LOCUS       Exported                4733 bp ds-DNA     circular SYN 20-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4733)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Wednesday, Mar 20, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4733

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /label=CMV enhancer

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        365..568

                     /label=CMV promoter

                     /note="human cytomegalovirus (CMV) immediate early

                     promoter"

     primer_bind     519..539

                     /label=CMV F

     misc_feature    591..671

                     /label=MCS

                     /note="multiple cloning site"

     regulatory      673..682

                     /regulatory_class="other"

                     /note="vertebrate consensus sequence for strong initiation

                     of translation (Kozak, 1987)"

     CDS             679..1398

                     /codon_start=1

                     /product="enhanced GFP"

                     /label=EGFP

                     /note="mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     primer_bind     complement(724..745)

                     /label=EGFP-N

     polyA_signal    1521..1642

                     /label=SV40 poly(A) signal

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1649..2104)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        2131..2235

                     /gene="bla"

                     /label=AmpR promoter

     promoter        2237..2594

                     /label=SV40 promoter

                     /note="SV40 enhancer and early promoter"

     rep_origin      2445..2580

                     /label=SV40 ori

                     /note="SV40 origin of replication"

     CDS             2629..3423

                     /codon_start=1

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

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /label=NeoR/KanR

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

                     (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3655..3702

                     /label=HSV TK poly(A) signal

                     /note="herpes simplex virus thymidine kinase

                     polyadenylation signal (Cole and Stacy, 1985)"

     rep_origin      4031..4619

                     /direction=RIGHT

                     /label=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 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc

      721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc

      781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg

      841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc

      901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc

      961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag

     1021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac

     1081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg

     1141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac

     1201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg

     1261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag

     1321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg

     1381 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg

     1441 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa

     1501 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca

     1561 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt

     1621 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc

     1681 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc

     1741 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag

     1801 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc

     1861 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa

     1921 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg

     1981 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt

     2041 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc

     2101 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa

     2161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat

     2221 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt

     2281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt

     2341 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc

     2401 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc

     2461 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg

     2521 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc

     2581 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa

     2641 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg

     2701 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc

     2761 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca

     2821 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc

     2881 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca

     2941 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat

     3001 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca

     3061 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg

     3121 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc

     3181 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct

     3241 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct

     3301 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac

     3361 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc

     3421 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag

     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg

     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg

     3601 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa

     3661 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt

     3721 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg

     3781 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca

     3841 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt

     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga

     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca

     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac

     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg

     4141 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag

     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac

     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt

     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg

     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc

     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc

     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc

     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa

     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt

     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat

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