质粒名称：pDsRed-Monomer-N1
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& |8 l* X! j) b5 s描述：pDsRed-Monomer-N1 is a mammalian expression vector that encodes DsRed-Monomer (DsRed.M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1). DsRed-Monomer contains forty-five amino acid substitutions (listed on page 2). When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection (DsRed-Monomer excitation and emission maxima = 557 nm and 585 nm, respectively). The DsRed-Monomer coding sequence is human codon-optimized for high expression in mammalian cells (2).
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DsRed-Monomer is well suited for use as a fusion tag. The multiple cloning site (MCS) in pDsRed-Monomer-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Monomer coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Monomer if they are in the same reading frame as DsRed-Monomer and there are no intervening stop codons. A Kozak consensus sequence is located immediately upstream of the DsRed-Monomer gene to enhance translational efficiency in eukaryotic systems (3). SV40 polyadenylation signals downstream of the DsRed-Monomer gene direct proper processing of the 3' end of the DsRed-Monomer mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in
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E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette confers kanamycin resistance in E. coli.
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用途：
: Y, k/ B3 Z6 T6 A' z. A: j: |- aretains the fluorescent properties of the native DsRed-Monomer protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene must be cloned into pDsRed-Monomer-N1 so that it is in frame with the DsRed-Monomer coding sequence, with no intervening in-frame stop codons. The inserted gene must include an initiating ATG codon. Recombinant pDsRed-Monomer-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). pDsRed-Monomer-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Monomer.
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The DsRed1-N Sequencing Primer (Cat. No. 632387) can be used to sequence genes cloned adjacent to the 5' end of the DsRed-Monomer coding region.
1 Y2 J$ M, q5 l" I* QFor Western blotting, the Living Colors™ DsRed Polyclonal Antibody (Cat. No. 632397) can be used to recognize the DsRed-Monomer protein. However, to generate optimal results it may be necessary to use a higher concentration of antibody than recommended on the DsRed Polyclonal Antibody Product Analysis Certificate.
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位点特征：
0 u8 c, l7 C5 U* t/ V• Human cytomegalovirus (CMV) immediate early promoter: 1–589
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Enhancer region: 59–465; TATA box: 554–560
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Transcription start point: 583
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C→G mutation to remove Sac I site: 569
2 `3 J4 Z7 p4 k+ r+ O3 u& B% X5 |9 P• Multiple Cloning Site: 591–671
6 L% O; q+ B5 X: Y$ Z) b- M• Human codon-optimized DsRed-Monomer gene
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Kozak consensus translation initiation site: 672–682
! T% V( b) x+ b7 C3 RStart codon (ATG): 679–681; Stop codon: 1354–1356
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Amino acid substitutions (DsRed→DsRed-Monomer)
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GCC→GAC (Ala-2 to Asp) mutation: 682–684
: h, F. ^- d, H- |, m4 ATCC→AAC (Ser-3 to Asn) mutation: 685–687
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TCC→ACC (Ser-4 to Thr) mutation: 688–690
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AAG→GAG (Lys-5 to Glu) mutation: 691–693
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AAC→GAC (Asn-6 to Asp) mutation: 694–696
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CGC→CAG (Arg-13 to Gln) mutation: 715–717
4 z! X. @1 n; y1 I0 T- eACC→TCC (Thr-21 to Ser) mutation: 739–741
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GAG→TAC (Glu-26 to Tyr) mutation: 754–756
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CGC→AAG (Arg-36 to Lys) mutation: 784–786
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CAC→ACC (His-41 to Thr) mutation: 799–801
8 _, t0 X2 ^3 e# p8 mAAC→CAG (Asn-42 to Gln) mutation: 802–804
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GTG→GCC (Val-44 to Ala) mutation: 808–810
& p/ m; n+ F- W+ i+ rAAG→CAG (Lys-47 to Gln) mutation: 817–819
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GTG→GCC (Val-71to Ala) mutation: 889–891
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AAG→ATG (Lys-83 to Met) mutation: 925–927
+ B6 \$ \2 i6 Y& AAAG→ACC (Lys-92 to Thr) mutation: 952–954
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GTG→TCC (Val-96 to Ser) mutation: 964–966
" m& G; F R$ p* y* L) u) F$ Y5 c" ~7 IACC→GAG (Thr-106 to Glu) mutation: 994–996
1 Z- D+ o4 Q; Q: I! J% v* m/ jACC→CAG (Thr-108 to Gln) mutation: 1000–1002
; a& t% P2 D) I, t( Q% V9 lTCC→ACC (Ser-117 to Thr) mutation: 1027–1029
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ATC→AAG (Ile-125 to Lys) mutation: 1051–1053
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TCC→GCC (Ser-131 to Ala) mutation: 1069–1071
( r" Q$ n l8 L, q- f6 hATG→GCC (Met-141 to Ala) mutation: 1099–1101
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GCC→CCC (Ala-145 to Pro) mutation: 1111–1113
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CGC→AAG (Arg-149 to Lys) mutation: 1123–1125
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CGC→CAG (Arg-153 to Gln) mutation: 1135–1137
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CAC→TCC (His-162 to Ser) mutation: 1162–1164
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AAG→CAC (Lys-163 to His) mutation: 1165–1167
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CTG→ACC (Leu-174 to Thr) mutation: 1198–1200
4 c5 D D% L2 t: _GTG→TGC (Val-175 to Cys) mutation: 1201–1203
; H# `' X, r! ^* G1 M( PGAG→GAC (Glu-176 to Asp) mutation: 1204–1206
$ w) ]# p) V; t3 z7 OTCC→ACC (Ser-179 to Thr) mutation: 1213–1215
1 F. A9 i. |6 a5 m+ n) QATC→GTG (Ile-180 to Val) mutation: 1216–1218
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ATG→AAG (Met-182 to Lys) mutation: 1222–1224
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TAC→AAC (Tyr-192 to Asn) mutation: 1252–1254
( W7 o, Z/ |$ `5 c( KProtocol No. PT3795-5
www.clontech.com Clontech Laboratories, Inc.
8 A* y% `& V( n- n# w+ d2 WVersion No. PR651711
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pDsRed-Monomer-N1 Vector Information
, b& I3 B5 f" fTAC→CAC (Tyr-193 to His) mutation: 1255–1257
8 G# @0 Y) R8 G& |9 ?& G, J& H3 `TCC→AAC (Ser-203 to Asn) mutation: 1285–1287
; ~$ u) K& x. o9 c6 }ATC→GTG (Ile-210 to Val) mutation: 1306–1308
$ L0 {/ B- d w7 \CGC→CAC (Arg-216 to His) mutation: 1324–1326
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ACC→GCC (Thr-217 to Ala) mutation: 1327–1329
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GGC→GCC (Gly-219 to Ala) mutation: 1333–1335
- [$ q$ _' w; v0 y- m- [' ICAC→TCC (His-222 to Ser) mutation: 1342–1344
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CTG→GGC (Leu-223 to Gly) mutation: 1345–1347
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TTC→TCC (Phe-224 to Ser) mutation: 1348–1350
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/ Q' a1 S1 Z0 U& g( F• SV40 early mRNA polyadenylation signal
) Q+ q: C* m2 Y( APolyadenylation signals: 1510–1515 & 1539–1544; mRNA 3' ends: 1548 & 1560
% m5 w Z1 }' J7 q; Y0 I; b- S• f1 single-strand DNA origin: 1607–2062 (Packages the noncoding strand of DsRed-Monomer)
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• Bacterial promoter for expression of Kanr gene:
9 S1 |2 n( F& j7 Y–35 region: 2124–2129; –10 region: 2147–2152
' F# k% y0 g" C8 D- n v1 }Transcription start point: 2159
' R# }1 q1 b; s• SV40 origin of replication: 2403–2538
2 R$ e, B$ C# T1 T5 b• SV40 early promoter
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Enhancer (72-bp tandem repeats): 2236–2307 & 2308–2379
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21-bp repeats: 2383–2403, 2404–2424 & 2426–2446
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Early promoter element: 2459–2465
$ R) E! s1 X7 _3 D1 i5 A4 L6 LMajor transcription start points: 2455, 2493, 2499 & 2504
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• Kanamycin/neomycin resistance gene
8 l6 ]6 e: o) h& DNeomycin phosphotransferase coding sequences: Start codon (ATG): 2587–2589; Stop codon: 3379–3381
' d& }6 H2 L- ?6 l7 ZG→A mutation to remove Pst I site: 2769
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C→A (Arg to Ser) mutation to remove BssH II site: 3115
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• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
2 t* X( U1 n t/ u% F% Z& |Polyadenylation signals: 3617–3622 & 3630–3635
* n1 J/ {: L. x4 s6 B• pUC plasmid replication origin: 3966–4609
6 h) m: d; d& z M2 i$ K% NSequencing primer location
( U0 A3 R( @+ }" W, H• DsRed1-N Sequencing Primer (Cat. No. 632387; 5'-GTACTGGAACTGGGGGGACAG-3'): 879–859
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Propagation in E. coli
. @" O, k) y) r1 T* M- X( S, |• Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires
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a host containing an F plasmid, such as the JM109 or XL1-Blue strains..
. o* V' B% A) Y% M+ F7 d$ Q' ^• Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
$ s, M5 R) j0 f; Y1 X1 a* M' P• E. coli replication origin: pUC
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• Copy number: high
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• Plasmid incompatibility group: pMB1/ColE1
! L0 {- P( v) ]/ k- j6 L, C; E) GExcitation and emission maxima of DsRed-Monomer
& a& N. }8 x5 r P• Excitation maximum = 557 nm
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• Emission maximum = 585
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* {/ Z% k! U# `4 T/ B" Y; C. P0 }$ v% y图谱：
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本帖最后由 小宝 于 08-7-23 11:54 编辑 ]
