• No results found

pselect-gfp-lc3 A mammalian expression plasmid containing the human LC3B gene fused at 5 end to the GFP gene

N/A
N/A
Protected

Academic year: 2021

Share "pselect-gfp-lc3 A mammalian expression plasmid containing the human LC3B gene fused at 5 end to the GFP gene"

Copied!
5
0
0

Loading.... (view fulltext now)

Full text

(1)

pSELECT-GFP-LC3

A mammalian expression plasmid containing the human LC3B gene fused at 5’end to the GFP gene

Catalog # psetz-gfplc3

For research use only

Version # 10K02-MM

ProduCt inForMAtion

Content:

- 20 µg of pSELECt-GFP-LC3plasmid provided as lyophilized DNA - 4 pouches of E. coliFast-Media®Zeo (2 TB and 2 Agar)

Storage and Stability:

Product is shipped at room temperature.

Lyophilized DNA should be resuspended upon receipt and stored at - 20˚C. Lyophilized DNA is stable 3 months at -20˚C. Resuspended DNA is stable more than one year at -20˚C.

Store E. coliFast-Media®Zeo at room temperature. Fast-Media® pouches are stable 18 months when stored properly.

Quality control:

Plasmid construct has been confirmed by restriction analysis and sequencing. Plasmid DNA was purified by ion exchange chromatography and lyophilized.

GEnErAL ProduCt uSE

pSELECT plasmids are specifically designed for strong and constitutive expression of a gene of interest in a wide variety of cell lines. They allow the selection of stable transfectants and offer a variety of selectable markers. pSELECT plasmids contain two expression cassettes: the first drives the expression of the gene of interest and the second drives the expression of a large choice of dominant selectable markers for both E. coli and mammalian cells. They are both terminating with a strong polyadenylation signal (polyA) that separates the two expression cassettes thus preventing any transcription interference. The late SV40 polyA terminates the transcription of the gene of interest while the human ß-globin polyA terminates the transcription of the selectable marker.

pSELECT-GFP-LC3 is a mammalian expression vector containing the human LC3B gene fused at its 5’ end to the green fluorescent protein (GFP) gene. This plasmid is selectable in bacteria and mammalian cells using Zeocin™. Expression of the GFP-LC3 fusion gene allows to visualize autophagosome formation in real time in live cells. During autophagosome formation, GFP-LC3 is processed and recruited to the autophagosome membrane, where it can be imaged as cytoplasmic puncta by high resolution fluorescence microscopy. The percentage of GFP-LC3 positive cells can be determined and is indicative of autophagosome formation. The same plasmid is available with the GFP gene alone as a control. This control plasmid is called pSELECT-NGFP-zeo (cat. code: psetz-ngfp). This plasmid is selectable in bacteria and mammalian cells with Zeocin™.

PLASMid FEAturES

First expression cassette

• hEF1-htLV prom is a composite promoter comprising the Elongation Factor-1a(EF-1a) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1apromoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1acore promoter to enhance stability of RNA.

• GFP::LC3Bfusion gene was generated by fusing a GFP variant to the 5’ end of the human LC3B gene. A synthetic intron was added between both moieties to increase the activity of GFP. This hybrid protein absorbs blue light (major peak at 480 nm) and emits green light (major peak at 505 nm). • SV40 pAn:the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.

• ori:a minimal E. coliorigin of replication to limit vector size, but with the same activity as the longer Ori.

Second expression cassette

• CMV enh/prom:The human cytomegalovirus immediate-early gene 1 promoter/enhancer was originally isolated from the Towne strain and was found to be stronger than any other viral promoters.

• EM7is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.

• Zeo: Resistance to Zeocin™ is conferred by the Sh ble gene from Streptoalloteichus hindustanusThe Sh blegene is driven by the CMV enhancer/promoter in tandem with the bacterial EM7 promoter allowing selection in both mammalian cells and E. coli.

ßGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allows efficient arrest of the transgene transcription4.

MEthodS

Plasmid resuspension:

Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1 µg/µl, resuspend the DNA in 20 µl of sterile H2O. Store resuspended plasmid at -20°C.

Selection of bacteria with E. coli Fast-Media®

Fast-Media®is a fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. Fast-Media® is a TB (liquid) or LB (solid) based medium that already contains the antibiotic. Fast-Media®Zeo can be ordered separately (cat. code: fas-zn-l (liquid), cat. code: fas-zn-s (solid)).

Method:

1- Pour the contents of a Fast-Media®pouch into a clean borosilicate glass bottle or flask.

2- Add 200 ml of distilled water to the flask

3- Heat in a microwave on MEDIUM power setting (about 400Watts), until bubbles start appearing (approximately 3 minutes). do not heat a closed container. do not autoclave Fast-Media®.

4- Swirl gently to mix the preparation. Be careful, the bottle and media are hot, use heatproof pads or gloves and care when handling. 5- Reheat the media for 30 seconds and gently swirl again. Repeat as necessary to completely dissolve the powder into solution. But be careful to avoid overboiling and volume loss.

6- Let agar medium cool to 45˚C before pouring plates. Let liquid media cool to 37˚C before seeding bacteria.

Note: Do not reheat solidified Fast-Media® as the antibiotic will be permanently destroyed by the procedure.

TECHNICAL SUPPORT Toll free (US): 888-457-5873 Outside US: (+1) 858-457-5873 Europe: +33 562-71-69-39 E-mail: info@invivogen.com Website: www.invivogen.com

3950 Sorrento Valley Blvd. Suite 100 San Diego, CA 92121 - USA

(2)

pSELECT-GFP-LC3

(4271 bp)

SV40 pAn

ßGlo pAn

Sh ble

pMB1 ori

hEF1/HTLV prom

EM7

hCMV enh/prom

GFP-hLC3b

NotI (-1)

SgfI (171)

HindIII (410)

AgeI (717)

BspHI (725)

Acc65I (944)

BamHI (1447)

BbsI (1464)

StuI (1682)

XbaI (1852)

NheI (1861)

EcoRI (2095)

SspI (2334)

SmaI (2790)

AseI (2938)

SpeI (3093)

SnaBI (3221)

NdeI (3326)

SdaI (3504)

PstI (3505)

PacI (3512)

BspLU11I (3522)

PacI (4252)

(3)

GCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATAC GCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAG AACATTTCTCTATCGAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC CCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACG GGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGT GATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAA CGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAG GGGCTCGCATCTCTCCTTCACGCG CCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCC TGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAG CCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTT

CTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGTCATCATGAGCAAGGGAG AAGAACTCTTTACTGGTGTTGTCCCAATTCTGGTTGAGCTGGATGGTGATGTGAATGGCCACAAATTCTCTGTG TCTGGTGAAGGTGAAGGAGATGCAACTTATGGAAAGCTGACTCTGAAGTTCATTTGTACAACAGGAAAGCTGCC AGTGCCTTGGCCAACTCTGGTGACCACCCTGACTTATGGTGTTCAATGTTTCAGCAGGTACCCTGACCACATGA AGCAGCATGACTTCTTTAAATCTGCAATGCCAGAAGGTTATGTTCAGGAGAGGACAATCTTCTTTAAGGATGAT GGAAATTATAAGACAAGGGCAGAAGTGAAGTTTGAAGGTGATACACTGGTTAACAGAATTGAGCTGAAAGGCAT TGATTTTAAGGAAGATGGAAACATTCTGGGTCACAAGCTGGAGTACAACTATAATTCTCACAATGTTTACATTA TGGCAGATAAGCAGAGGAATGGAATTAAGGCTAATTTCAAGATTAGACACAACATTGAGGATGGATCTGTCCAA CTGGCAGACCATTACCAGCAGAACACCCCTATTGGTGATGGCCCAGTTCTCCTCCCAGATAATCACTATCTCAG CACTCAATCTGCTCTGTCCAAAGACCCTAATGAGAAAAGAGACCACATGGTCCTCCTGGAGTTTGTGACAGCAG CAGGAATTACTCTGGGAATGGATGAGCTGTACAAGGGAGGTGGATCCATGCCGTCGGAGAAGACCTTCAAGCAG CGCCGCACCTTCGAACAAAGAGTAGAAGATGTCCGACTTATTCGAGAGCAGCATCCAACCAAAATCCCGGTGAT AATAGAACGATACAAGGGTGAGAAGCAGCTTCCTGTTCTGGATAAAACAAAGTTCCTTGTACCTGACCATGTCA ACATGAGTGAGCTCATCAAGATAATTAGAAGGCGCTTACAGCTCAATGCTAATCAGGCCTTCTTCCTGTTGGTG AACGGACACAGCATGGTCAGCGTCTCCACACCAATCTCAGAGGTGTATGAGAGTGAGAAAGATGAAGATGGATT CCTGTACATGGTCTATGCCTCCCAGGAGACGTTCGGGATGAAATTGTCAGTGTAAAACCAGAAAAAATGCAGCT CTTCTAGAATTGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACA Me tSer Ly sGl yG l uGl uLeuPheThr Gl yVa l Va l P ro I l eLeuVa l Gl uLeuAspGl yAspVa l AsnGl yHi s Ly sPheSer Va l Ser Gl yGl uGl yGl uGl yAspAl aThr T y rGl yLy sLeuThr LeuLy sPhe I l eCysThr Thr Gl yLy sLeuP r

oVa l P roT rpP roThr LeuVa l Thr Thr LeuThr T y rGl yVa l Gl nCysPheSer A rgT y rP roAspHi s Me tL y sGl nHi sAspPhePheLy sSer Al aMe tP roGl uGl y T y rVa l Gl nGl uArgThr I l ePhePheLysAspAsp Gl yAsnT y rLy sThr A rgAl aGl uVa l Ly sPheGl uGl yAspThr LeuVa l AsnArg I l eGl uLeuLy sGl y I l eAspPheLy sGl uAspGl yAsn I l eLeuGl yHi s Ly sLeuGl uTyrAsnTyrAsnSer Hi sAsnVa l T y r I l eM e tAl aAspLy sGl nArgAsnGl y I l eLysAl aAsnPheLy s I l eArgHi sAsn I l eGl uAspGl ySer Va l Gl n LeuAl aAspHi s T y rGl nGl nAsnThr P ro I l eGl yAspGl y P roVa l LeuLeuP roAspAsnHi s T y rLeuSe r Thr Gl nSer Al aLeuSer LysAspP roAsnGl uLysArgAspHi s Me tVa l LeuLeuGl uPheVa l Thr Al aA

l aGl y I l eThr LeuGl yMe tAspGl uLeuT y rLy sGl yGl yGl ySer Me tP roSer Gl uLy sThr PheLy sGl n A rgArgThr PheGl uGl nArgVa l Gl uAspVa l A rgLeu I l eArgGl uGl nHi s P roThr Ly s I l eP roVa l I l e I l eGl uArgT y rLy sGl yGl uLy sGl nLeuP roVa l LeuAspLy sThr Ly sPheLeuVa l P roAspHi s Va l A

s nMe tSer Gl uLeu I l eLy s I l e I l eArgArgArgLeuGl nLeuAsnAl aAsnGl nAl aPhePheLeuLeuVa l AsnGl yHi s Ser Me tVa l Ser Va l Ser Thr P ro I l eSer Gl uVa l T y rGl uSer Gl uLysAspGl uAspGl yPh eLeuT y r Me tVa l TyrAl aSer Gl nGl uThr PheGl yMe tLy sLeuSer Va l •••

NotI (-1)

SgfI (171)

HindIII (410)

AgeI (717)

BspHI (725)

Acc65I (944)

BamHI (1447)

BbsI (1464)

StuI (1682)

XbaI (1852)

NheI (1861)

1 7 5 149 223 297 371 445 519 593 667 741 815 889 963 1037 1111 1185 1259 1333 1407 1481 1555 1629 1703 1777 1851 1925 1 5 3 0 5 4 7 9 104 128 153 178 202 227 252 276 301 326 350

(4)

AGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGT AAAACCTCTACAAATGTGGTATGGAATTCTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTA CTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCA GCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTAT GTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAA TGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTT GGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTAGCTTATCCTCAGTCC TGCTCCTCTGCCACAAAGTGCACGCAGTTGCCGGCCGGGTCGCGCAGGGCGAACTCCCGCCCCCACGGCTGCTC GCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGACCTCCGACCACTCGGCGTACA GCTCGTCCAGGCCGCGCACCCACACCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCCTGGACCGCGCTGATG AACAGGGTCACGTCGTCCCGGACCACACCGGCGAAGTCGTCCTCCACGAAGTCCCGGGAGAACCCGAGCCGGTC GGTCCAGAACTCGACCGCTCCGGCGACGTCGCGCGCGGTGAGCACCGGAACGGCACTGGTCAACTTGGCCATG A TGGCCCTCCTATAGTGAGTCGTATTATACTATGCCGATATACTATGCCGATGATTAATTGTCAAAACAGCGTGG ATGGCGTCTCCAGC T TATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTA CCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTA C T A G TCAAAACA AACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATG TACTGCCAAAACCGCATCAT CATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCA TAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCA TATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTC CCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGG CGGGCCATTTACCGTAAGTTATGTAACGC C T G C AG G TT AA TT AAGAACATGTGAGCAAAAGGCCAGCAAAA GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT •••AspG l nGl uGl uAl aVa l PheHi s Va l CysAsnGl yA l aP roAspArgLeuAl aPheGl uArgGl y T rpP roGl nGl u Gl y I l eGl uThr Me tAl aP roGl ySer Al aAspArgPheAsnThr Ser Va l Va l Gl uSer T rpGl uAl aT y rLe uGl uAspLeuGl yA rgVa l T rpVa l T rpAl aLeuThr A snAspP roVa l Va l Gl nAspGl nVa l A l aSer I l eP heLeuThr Va l AspAspArgVa l Va l Gl yA l aPheAspAspGl uVa l PheAspArgSer PheGl yLeuArgAsp Thr T rpPheGl uVa l A l aGl yA l aVa l AspArgAl aThr LeuVa l P roVa l A l aSer Thr LeuLysAl aMe t

EcoRI (2095)

SspI (2334)

SmaI (2790)

AseI (2938)

SpeI (3093)

SnaBI (3221)

NdeI (3326)

SdaI (3504)

PstI (3505)

PacI (3512)

BspLU11I (3522)

1999 2073 2147 2221 2295 2369 2443 2517 2591 2665 2739 2813 2887 2961 3034 3107 3181 3255 3329 3403 3477 3548 3622 3696 3770 3844 3918 125 122 9 8 7 3 4 8 2 4

(5)

GAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG GAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTAACATTTAAATC A

PacI (4252)

3992 4066 4140 4214

References

Related documents

Since the crack geometry is symmetric, 1/4 cylinder model with an inner semi-elliptic surface crack is assumed for finite element analysis, as shown in Fig..

These characteristics will be an eye opener for those who are using JIT system that low inventory, flexible production, good ationship with supplier and high

From this table one can observe that in the deposition of Ti and Cu onto graphene surface, the interaction of these two metal atoms with HOMO of graphene predominates , and

The proposed model combines SNAuth-SPMAODV Routing with Wireless Transport Layer Security (WTLS) to defend against Denial of Service (DoS) attack and it also provides

Fast Radial and Bilateral Symmetry Detection Using Inverted Gradient Hash MapsR.

is no KAP study on AIDS documented so far in Malaysia for lower secondary schooL The purpose of the study, therefore, was to determine the knowledge, attitude

Since section 3004 has been amended to extend corrective action re- quirements to all solid waste management units at facilities seeking a RCRA permit, the

After briefly comparing the benefits and negatives associated with two forms of electronic com- merce, electronic data interchange ("EDI") and forms