src (dom. neg.) in pUSE amp, cDNA (K296R/Y528F) - Millipore

src (dom. neg.) in pUSE amp, cDNA 

(K296R/Y528F) 

7.45 kb 

Catalog # 21-154 

Lot # 17759 

Product Description: Transfection grade eukaryotic 

expression vector containing mouse src (dom. neg.) 

under the control of the CMV promoter as illustrated. 

The cDNA was inserted as an Hind III fragment into 

the Hind III site of pUSEamp(+) multiple cloning site 

(Catalog # 21-147). 

The dominant negative src cDNA was derived from 

the neuronal form of c-src subjected to site directed 

mutagenesis to delete neuronal specific sequences. 

The cDNA contains two point mutations, the 

substitution of arginine for lysine at residue 296 and 

phenylalanine for tyrosine at residue 528. NOTE: 

These residues correspond to K295 and Y527 in avian 

src. 

Formulation: 50mg affinity purified DNA eluted and 

packaged aseptically in 100ml 10mM Tris-HCl, pH 7.4, 

1mM EDTA. Frozen solution. 

Storage and Stability: Stable for 6 months at -20ºC 

from date of shipment. 

FOR RESEARCH USE ONLY 

NOT FOR USE IN HUMANS 

Quality Control Testing 

Diagnostic DNA digest: This lot of DNA was cut with 

Hind III, which generated the expected fragments of 

approximately 1.9 and 5.5Kb. 

Purity: The OD260/OD280 for this lot of cDNA is 1.72. 

Expression in Transient Transfection Assay: 

Expression of src was detected by immunoblotting of 

src (dom. neg.) in pUSEamp transfected Cos-1 cells 

with 1μg/ml of anti-src (Catalog # 05-184). 

Immunoblot Analysis: 

RIPA lysates of transiently transfected Cos- 

1 cells were resolved by electrophoresis, 

transferred to nitrocellulose and probed with 

anti-src (Catalog #05-184, 1μg/ml). 

Proteins were visualized using a goat antimouse 

secondary antibody conjugated to 

HRP and a chemiluminescence detection 

system. Lane 1, pUSE src (dom. neg.) 

transfected cells; Lane 2 untransfected 

control. Arrow indicates src. 

Background: Src is the prototypical non-receptor protein tyrosine kinase whose activity is negatively regulated by 

phosphorylation of a C-terminal tyrosine residue by the tyrosine kinase Csk (C-terminal Src kinase). Src is involved in 

multiple signaling pathways, which include integrin mediated cell adhesion and growth factor initiated cell proliferation. 

The substitution of arginine for lysine (residue 296) in the ATP binding pocket of the tyrosine kinase domain abolishes 

phosphotransferase activity. The phenylalanine substitution for tyrosine (residue 528) abolishes phosphorylation and 

thereby prevents intramolecular interaction between the C-terminus and SH2 domain of Src. This kinase inactive src 

mutant functions as a dominant negative and renders the SH2 domain, and probably the SH3 domain, accessible to 

cellular binding proteins that normally contribute to activation of Src.

Page Two of Three 


Lot 17759 

Application Reference: 

Mukhopadhyay, D., et al., Nature 375: 577-581, 1995. 

References: 

Zang, Q., et al., J. Biol. Chem. 272: 13275-13280, 1997. 

Brown, M.T., et al., Biochim. Biophys. Acta 1287: 121-149, 1996. 

Barone, M.V., et al., Nature 378: 509-512, 1996. 

Plasmid Information: 

Multiple Cloning Site of pUSE (+) [5’?3’]: 

Nhe I 

Pme I 

Afl II 

Hind III 

Asp718 I 

Kpn I 

BamH I 

BstX I 

EcoR I 

EcoR V 

BstX I 

Not I 

Xho I 

Xba I 

Apa I 

Locations of cDNA Features: 

cDNA insert: nt 912-2935 

src ORF: nt 1079-2686 

K296R: AAA to AGG nt 1967-1969 

Y528F: TAC to TCC nt 2663-2665 

PmeI 

Schematic Map of src (dom. neg.) cDNA : 

DNA Sequence: 

The complete sequence file of this plasmid is available 

at: www.upstatebiotech.com 

Transient transfection protocol for Cos-1 cells 

Note: The protocol described below has been optimized for Cos-1 cells. Successful transfection of each cell type 

requires optimization of the basic protocol. Variables to consider for optimization include, but are not limited to: cell 

density, transfection reagent, duration of transfection and DNA concentration. 

1. Seed a six-well tissue culture plate with 1-2 x 10 5 Cos-1 cells in 2ml of DMEM supplemented with 10% fetal calf 

serum. Incubate for 18-24 hours at 37ºC or until the cells are 40-60% confluent in a CO2 humidified incubator. 

2. Dilute 1-2μg of DNA into 100μl serum free DMEM in a sterile tube. To this tube, add 6μl of PLUS Reagent 

found in the LIPOFECTAMINE PLUS Reagent kit (Life Technologies). Allow mixture to stand for 15 minutes 

at room temperature. In a second tube dilute 4μl of LIPOFECTAMINE Reagent into 100μl serum free DMEM. 

3. Combine the two solutions, mixing gently and incubate at room temperature for 10-15 minutes. 

4. Wash cells once with 2ml serum free DMEM. 

5. For each transfection, add 0.8ml serum-free medium to each tube containing the LIPOFECTAMINE PLUS 

Reagent - DNA complexes. Mix gently and overlay the complexes onto cells. 

6. Incubate the cells for 3 hours at 37ºC in a CO2 humidified incubator. 

7. Remove the DNA-containing medium and replace with 2ml of DMEM supplemented with 10% fetal calf serum. 

Incubate cells 37ºC in a CO2 humidified incubator for an additional 48-72 hours. 

Hind III 

8. Assay cell extracts for gene expression or activity at 48-72 hours post transfection. 

LIPOFECTAMINE PLUS IS a registered trademark of Life Technologies, Inc. 

Xba I 

src ORF 

Hind iii

Page Three of Three 


Lot # 17759 

Restriction Endonuclease Map by Position: src (dom. neg.) in pUSEamp 

(Frequency < 3; Recognition sites >5 bp) 

Restriction sites found in insert are in bold type. 

12 Bgl II 

12 NcrI 

55 StySJ 

161 MfeI 

161 Mun I 

180 Bpu10 I 

208 Nru I 

228 Afl III 

228 Mlu I 

249 Spe I 

484 Nde I 

590 BsaA I 

590 Eco105 I 

590 SnaB I 

593 Eco105 I 

876 Bsa I 

876 Eco31I 

895 Nhe I 

899 Ace II 

904 Pme I 

908 Afl II 

908 Bfr I 

908 Bst98 I 

911 Bbr I 

911 EcoVIII 

911 Hind III 

913 Bfr I 

918 BspDI 

918 Cla I 

961 EcoB I 

1031 Xba I 

1053 Xcm I 

1085 Bsg I 

1217 Mlu113 I 

1219 Sac II 

1220 Bsp120 I 

1224 Apa I 

1224 PpeI 

1246 Mlu113 I 

1248 Eco52 I 

1248 Sac II 

1248 Xma III 

1289 Tth111 I 

1328 UbaEI 

1330 BstE II 

1330 EcaI 

1342 BstX I 

1360 EclHK I 

1469 Age I 

1469 PinAI 

1489 Xcm I 

1518 Bpu10 I 

1593 PpuM I 

1610 Bsa I 

1610 Eco31I 

1721 Bsa XI 

1837 RleA I 

1846 Van91 I 

1935 PpuM I 

2000 Stu I 

2009 EcoN I 

2010 Sse8387 I 

2022 BspH I 

2041 StySJ 

2051 Ecoprr I 

2072 Bsg I 

2153 Ssp I 

2173 Van91 I 

2178 Afl III 

2178 BspLU11 I 

2201 CfrA I 

2239 PpuM I 

2274 SexA I 

2325 Bbs I 

2364 Van91 I 

2484 EcoRD3 

2498 EcoRD3 

2502 SexA I 

2622 Sse8387 I 

2624 Stu I 

2640 Bbs I 

2697 Fsp I 

2724 Bcg I' 

2739 UbaEI 

2758 Bcg I 

2809 Xmn I 

2872 Xcm I 

2890 EcoR I 

2890 HalI 

2890 SsoI 

2908 AccEBI 

2908 BamH I 

2908 BnaI 

2914 Xba I 

2934 Bbr I 

2934 EcoVIII 

2934 Hind III 

2940 Acc65 I 

2940 Asp718 I 

2944 Kpn I 

2945 Asp718 I 

2952 AccEBI 

2952 BamH I 

2952 BnaI 

2958 Spe I 

2971 BstX I 

2975 EcoR I 

2975 HalI 

2975 SsoI 

2984 M.SmaDam 

2987 EcoR V 

2997 BstX I 

3002 CciNI 

3002 Eco52 I 

3002 Not I 

3002 Xma III 

3007 BsuMI 

3007 Sex I 

3008 Ccr I 

3010 Sci I 

3013 Ccr I 

3014 Xba I 

3020 Bsp120 I 

3024 Apa I 

3024 PpeI 

3029 Pme I 

3037 BmeTI 

3038 Bcl I 

3060 EcoR124 I 

3240 Bbs I 

3374 Rsr II 

3446 NgoM I 

3448 Nae I 

3448 SauLPI 

3548 AdeI 

3551 BsaA I 

3554 Dra III 

3557 AdeI 

3608 Bsa XI 

3610 UbaD I 

3749 Xmn I 

3823 BstAPI 

3824 ApaB I 

3824 Ppu10 I 

3826 BfrB I 

3828 Nsi I 

3845 SexA I 

3895 BstAPI 

3896 ApaB I 

3896 Ppu10 I 

3898 BfrB I 

3900 Nsi I 

4077 Stu I 

4078 Avr II 

4128 BmeTI 

4129 Bcl I 

4147 BsaB I 

4147 Mam I 

4152 Mam I 

4194 Eco52 I 

4194 Xma III 

4390 Fsp I 

4406 Tth111 I 

4569 EcoR124 II 

4592 BsaA I 

4684 BscE I 

4684 BseP I 

4685 BssH II 

4690 BscE I 

4788 NgoM I 

4790 Nae I 

4790 SauLPI 

4804 Cpo I 

4804 Csp I 

4809 Cpo I 

4970 Cbi I 

4970 Csp45 I 

4970 Sfu I 

4974 Cbi I 

4974 Sfu I 

5071 NgoM I 

5073 Nae I 

5073 SauLPI 

5101 EcoD XXI 

5207 CfrA I 

5208 BsaM I 

5208 Bsm I 

5260 Bst1107 I 

5502 Bsa XI 

5519 StyLT III 

5639 Afl III 

5639 BspLU11 I 

5958 EcoRD2 

5971 EcoRD2 

6359 BspH I 

6532 EclHK I 

6593 Bsa I 

6593 Eco31I 

6754 Fsp I 

6758 Psp1406 I 

6902 Pvu I 

6902 Xor II 

6904 Xor II 

7012 Sca I 

7030 StyLT III 

7037 Bcg I' 

7071 Bcg I 

7131 Psp1406 I 

7131 Xmn I 

7189 EcoK I 

7189 StySP I 

7336 Ssp I 

7367 BspH I

src (dom. neg.) in pUSE amp, cDNA (K296R/Y528F) - Millipore

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