cytoplasmic EKAR (EGFP-mRFP) vector (V000047)

Basic Vector Information

      • Vector Name:
      • cytoplasmic EKAR (EGFP-mRFP)
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 5981 bp
      • Type:
      • Mammalian Expression
      • Copy Number:
      • High Copy
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • CMV Forward

cytoplasmic EKAR (EGFP-mRFP) vector Vector Map

cytoplasmic EKAR (EGFP-mRFP)5981 bp60012001800240030003600420048005400CMV enhancerCMV promoterchimeric intronT7 promotermRFP1EGFPSV40 poly(A) signalf1 oripRS-markerpGEX 3'pBRforEcoAmpR promoterAmpRori

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 ℃.

cytoplasmic EKAR (EGFP-mRFP) vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                5981 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    cytoplasmic EKAR (EGFP-mRFP)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  AUTHORS   Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, 
            Svoboda K.
  TITLE     A genetically encoded fluorescent sensor of ERK activity
  JOURNAL   PNAS 2008 Dec 9;105(49):19264-9
  PUBMED    19033456
REFERENCE   2  (bases 1 to 5981)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PNAS"; 
            date: "2008-12-9"; volume: "105(49)"; pages: "19264-"
FEATURES             Location/Qualifiers
     source          1..5981
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1058..1732
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(1188..1206)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1456..1475
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1650..1673
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2300..3016
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2300..3016
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2345..2366)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2606..2625)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2953..2974
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3095..3216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3160..3179
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3397..3852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3484..3503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3694..3715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3919..3938)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4038..4060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4098..4116)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4184..4288
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4289..5149
                     /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     complement(4507..4526)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5320..5908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5809..5828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"

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