pEBFP-N1 carries a blue fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP). The EBFP gene contains four amino acid substitutions. The Tyr-66 to His substitution gives EBFP fluorescence excitation and emission maxima (380 and 440 nm, respectively) similar to other blue emission variants (1–3). The other three substitutions (Phe-64 to Leu; Ser-65 to Thr; and Tyr- 145 to Phe) enhance the brightness and solubility of the protein, primarily due to improved proteinfolding properties and efficiency of chromophore formation (1, 4, 5). The Em of EBFP is 31,000 cm–1M–1 for 380-nm excitation, leading to a fluorescent signal that is 2–3-fold brighter than other blue variants of GFP and roughly equivalent to wt GFP. In addition, the rate of photobleaching of EBFP is one-half to one-third that of P4-3, a popular predecessor to EBFP (1). EBFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (6). Furthermore, upstream sequences flanking EBFP have been converted to a Kozak consensus translation initiation site (7). These changes increase the translational efficiency of the EBFP mRNA and consequently the expression of EBFP in mammalian and plant cells.The MCS in pEBFP-N1 is between the immediate early promoter of CMV (PCMV IE) and the EBFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EBFP if they are in the same reading frame as EBFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. EBFP with N-terminal fusion moieties retain the fluorescent properties of the native protein and thus can be used to localize fusion proteins in vivo.The vector contains an SV40 origin for replication and a neomycin resistance (Neor ) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
| 载体基本信息 | |||
| 载体名称 | pEBFP-N1 | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4733 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | 5d[CGTCGCCGTCCAGCTCGACCAG]3 |
pEBFP-N1 载体载体图谱
pEBFP-N1 载体载体序列
LOCUS Exported 4733 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4733)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4733
/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 591..671
/note="MCS"
/note="multiple cloning site"
CDS 679..1398
/codon_start=1
/product="enhanced blue variant of GFP"
/note="EBFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1521..1642
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1649..2104)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2131..2235
/gene="bla"
/note="AmpR promoter"
promoter 2237..2594
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2445..2580
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2629..3423
/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 3655..3702
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4031..4619
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
