Human EGFR ORF clone (NM_005228) (Cat. No.: V041306)

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Basic Information

Note: 2 NT, 1 AA Mismatches

Name:: pCMV-Neo-EGFR(human)-L858R-ECFP

Accession ID:: NM_005228.5

Antibiotic Resistance:: Kanamycin

Length:: 8339 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells

Selection Marker:: Neo/G418;ECFP

Copy Number:: High

Promoter:: CMV

5' Primer:: CMV-F

Fusion Tag:: ECFP

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

Expression Method:: Transient

Gene Synonyms:: ERBB; ERBB1; ERRP; HER1; mENA; NISBD2; PIG61

Transcript Definition:: Homo sapiens epidermal growth factor receptor (EGFR), transcript variant 1, mRNA

Cat. No.: V041306 pCMV-Neo-EGFR(human)-L858R-ECFP

$ 299.1

In stock, 1-2 weeks for quality controls

Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

Human EGFR ORF clone (NM_005228) (Cat. No.: V041306) Sequence

LOCUS       V041306                 8339 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V041306
VERSION     V041306
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        61..364
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        365..568
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             624..4253
                     /EC_number=""
                     /codon_start=1
                     /gene=""
                     /note=""
                     /product="precursor of human epidermal growth factor
                     receptor"
                     /protein_id=""
                     /transl_table=1
                     /translation="MRPSGTAGAALLALLAALCPASRA,LEEKKVCQGTSNKLTQLGTF
                     EDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLEN
                     LQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESI
                     QWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSG
                     RCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVN
                     PEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNG
                     IGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTV
                     KEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEIS
                     DGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCW
                     GPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRG
                     PDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLE
                     GCPTNGPKIPS,IATGMVGALLLLLVVALGIGLFM,RRRHIVRKRTLRRLLQERELVEP
                     LTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREA
                     TSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNI
                     GSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGRAKLLGAEEKE
                     YHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSIL
                     EKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDER
                     MHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNS
                     TVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGS
                     VQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSH
                     QISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA"
                     /label="EGFR"
     CDS             4287..5006
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="enhanced CFP"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNG
                     IKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK*"
                     /label="ECFP"
     polyA_signal    5127..5248
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(5255..5710)
                     /direction=LEFT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        5737..5841
                     /gene="bla"
                     /label="AmpR promoter"
     promoter        5843..6200
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6235..7029
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    7261..7308
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      7637..8225
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"