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ERroGFP_iE_pCDNA3 vector (V000494#)

Basic Vector Information

Basic Vector Information
Vector Name ERroGFP_iE_pCDNA3 Antibiotic Resistance Ampicillin
Length 6256 bp Type Mammalian Expression
Copy Number High Copy Cloning Method Restriction Enzyme
5' Primer CMV forward 3' Primer SP6

ERroGFP_iE_pCDNA3 vector Vector Map

ERroGFP_iE_pCDNA36256 bp30060090012001500180021002400270030003300360039004200450048005100540057006000pRS-markerCMV enhancerCMV promoterFLAGGFP-RGFP-FSP6 promoterBGH-revf1 oripBABE 3'NeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoter

Plasmid Resuspension 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 ℃.

ERroGFP_iE_pCDNA3 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                6256 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression vector encoding for the ER targeted and FLAGM1 
            tagged redox probe roGFPiE (roGFP C147, E147b, C204) .
ACCESSION   .
VERSION     .
KEYWORDS    ERroGFP_iE_pCDNA3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   Avezov E, Cross BC, Kaminski Schierle GS, Winters M, Harding HP, 
            Melo EP, Kaminski CF, Ron D
  TITLE     Lifetime imaging of a fluorescent protein sensor reveals surprising 
            stability of ER thiol redox.
  JOURNAL   J Cell Biol. 2013 Apr 15;201(2):337-49. doi: 10.1083/jcb.201211155.
  PUBMED    23589496
REFERENCE   2  (bases 1 to 6256)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1083/jcb"; 
            journalName: "J Cell Biol"; date: "2013-04-15- 15"; volume: "201"; 
            issue: "2"; pages: "337-49"
FEATURES             Location/Qualifiers
     source          1..6256
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     815..839
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             949..972
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1006..1034)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1632..1652
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(1808..1826)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1809..1826)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1846..1863)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1852..2076
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2122..2550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2209..2228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2419..2440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2559..2579)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2564..2894
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2745..2880
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2807..2826
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2961..3755
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3015..3034)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3625..3644
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3929..4050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3966..3985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4020..4039
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4112..4134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4123..4139
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4147..4177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4501..5089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4581..4600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5260..6120)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5883..5902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6121..6225)
                     /gene="bla"
                     /label=AmpR promoter

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