pECFP-N1质粒图谱及相关信息
质粒名称：pECFP-N1用途：The recombinant ECFP vector can be transfected into mammalian cells using any standard transfectionU a!t'B'Q!n*y
method. If required, stable transformants can be selected using G418 (8). pECFP-N1 can also be usedo`Hu"P/E
simply to express ECFP in a cell line of interest (e.g., as a transfection marker). ECFP can be used for]n#uz{ g:J
double-labeling experiments together with EYFP using standard fluorescence microscopy and the8t N F c Qu1O
appropriate filter sets.
Descriptionm:D`a3X9K]
pECFP-N1 encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent
protein gene (GFP). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp
substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm)
and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission
variants (1, 2). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153"E z w `6P*V6F
to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due to
improved protein folding properties and efficiency of chromophore formation (1, 3, 4). In addition tol](Q3`(m%M
the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame
comprised almost entirely of preferred human codons (5, 6). Furthermore, upstream sequences flanking){)M)SI#c
ECFP have been converted to a Kozak consensus translation initiation site (7). These changes increasey2Y D4C8g'p7bv
the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian
and plant cells.
The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection7q8K e'Inf)`N i r
(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. The MCS in pECFP-N1 is
between the immediate early promoter of CMV (PCMV IE) and the ECFP coding sequences. Genes cloned6giE1]g-wg
into the MCS will be expressed as fusions to the N-terminus of ECFP if they are in the same reading frame
as ECFP and there are no intervening stop codons. The inserted gene should include an initiating ATG+fT"c.J5U
codon. ECFP fusion proteins retain the fluorescent properties of the native protein in vivo.
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特征：
Location of features0a$eO/i%r+XM
• Human cytomegalovirus (CMV) immediate early promoter: 1–589
Enhancer region: 59–465j r0`v [K7dn
TATA box: 554–560
Transcription start point: 583
C→G mutation to remove Sac I site: 569|*zJ!L~*a J-[pd
• MCS: 591–671
• Enhanced cyan fluorescent protein (ECFP) gene
Kozak consensus translation initiation site: 672–682!l/o#li%pT |'G
Start codon (ATG): 679–681; stop codon: 1396–1398
Insertion of Val at position 2: 682–684
ECFP mutations:
Phe-64 to Leu, Ser-65 to Thr, and Tyr-66 to Trp: 871–879 W3Q&|2V"?%a
Asn-146 to Ile: 1117–1119$s | `D| ]%v ?|
Met-153 to Thr: 1138–1140d$X.um0P
Val-163 to Ala: 1168–1170a1t%\C"EB8S
His-231 to Leu mutation (A→T): 1373
• SV40 early mRNA polyadenylation signalGhm+O)k3]1h
Polyadenylation signals: 1552–1557 & 1581–1586
mRNA 3' ends: 1590 & 1602y l)z-r`i? k+w9P
• f1 single-strand DNA origin: 1649–2104Rglq ]9x(_ k)[A
(Packages the noncoding strand of ECFP.)5I/KS#^w'B1xT1P P
• Bacterial promoter for expression of Kanr gene:g#KeH,J&A U-E'O
–35 region: 2166–2171; –10 region: 2189–2194
Transcription start point: 2201 Gj.~ A v
• SV40 origin of replication: 2445–2580
• SV40 early promoterG Bo6\ A6n
Enhancer (72-bp tandem repeats): 2278–2349 & 2350–2421
21-bp repeats: 2425–2445, 2446–2466 & 2468–2488
Early promoter element: 2501–2507M} L0W*s8S8k
Major transcription start points: 2497, 2535, 2541 & 2546
• Kanamycin/neomycin resistance geneQ7^PM I/@].C K3Y
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2629–2631; stop codon: 3421–3423
G→A mutation to remove Pst I site: 2811W1XFU2\5xp
C→A (Arg to Ser) mutation to remove BssH II site: 31574[&rk7d1B ~[}
• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3659–3664 & 3672–3677%S zU A0QG6A`$u!O
• pUC plasmid replication origin: 4008–4651;kA6}8O7m oP%z [
Primer Locations
• EGFP-N Sequencing Primer (#6479-1): 745–724 ~J _ yMP1?7z
• EGFP-C Sequencing Primer (#6478-1): 1332–1353
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 JM101 or XL1-Blue.:] BFQ yg
• Selectable marker: plasmid confers resistance to kanamycin (30 μg/ml) in E. coli hosts.
• E. coli replication origin: pUC; Copy number: ~500J i7c@Jm
• Plasmid incompatibility group: pMB1/Col E18` ?$e b5~[!_ g
图谱：[attach]3335[/attach][align=center][/align]
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图谱附件：[attach]3334[/attach]
[[i] 本帖最后由 小宝 于 08-7-23 11:45 编辑 [/i]] 看了一下，正在用pEGFP-N作siRNA序列筛选，希望能的大理想的效果
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