pECFP- N1

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  • Model: PVT1218
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pECFP-N1

PVT1218         2ug

 

pECFP-N1 Information

Promoter: CMV promoter

Replicator: pUC ori, F1 ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation serial plasmids; lactating plasmids; lactation cyan plasmids

Plasmid size: 4733bp

Plasmid label: C-ECFP

Prokaryotic resistance: kanamycin Kan (50 mu g/ml)

Screening markers: neomycin Neo/G418

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 C, 5%CO2

Induction mode: no induction, instantaneous expression

5'sequencing primers: CMV-F (CGCAAATGGGCGGTAGGCGTG)

3'Sequencing Primer: Sv40-polyA-R (GAAATTTGATGCTATTGC)

 

 

pECFP-N1 Description
PECFP-N1 encodes Aequorea enhanced green fluorescent variant of Vitoria green fluorescent protein gene (GFP). The ECFP gene contains 6 amino acid substituents. Tyr-66 gives Trpsubstitution ECFP fluorescence excitation (the main peak at 433nm and the secondary peak at 453nm) and similar emission from other blue emission variants (the main peak of 475nm and the second peak of 501nm). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhanced the brightness and solubility of the protein, mainly due to the improved protein folding properties and the efficiency of the chromophore formation. In addition to the chromophore mutation, ECFP contains > 190 silent mutations, creating an open reading framework that is almost entirely the preferred human codon. In addition, the upstream sequences of ECFP flanks have been translated into Kozak consistent translation initiation sites. These changes increase the translation efficiency of ECFP mRNA, thereby increasing the expression of ECFP in mammalian and plant cells.

pECFP-N1 encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission variants (1, 2). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due to improved protein folding properties and efficiency of chromophore formation (1, 3, 4). In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (5, 6). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (7). These changes increase the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells. The vector contains an SV40 origin of replication and a neomycin resistance (Neor ) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. The MCS in pECFP-N1 is between the immediate early promoter of CMV (PCMV IE) and the ECFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of ECFP if they are in the same reading frame as ECFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. ECFP fusion proteins retain the fluorescent properties of the native protein in vivo. The recombinant ECFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (8). pECFP-N1 can also be used simply to express ECFP in a cell line of interest (e.g., as a transfection marker). ECFP can be used for double-labeling experiments together with EYFP using standard fluorescence microscopy and the appropriate filter sets.

pECFP-N1-vector

 

pECFP-N1 Sequence

LOCUS       Exported                4733 bp ds-DNA     circular SYN 18-JAN-2018

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pECFP-N1

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4733)

  AUTHORS   .

  TITLE     Direct Submission

FEATURES             Location/Qualifiers

     source          1..4733

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /note="CMV enhancer"

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        365..568

                     /note="CMV promoter"

                     /note="human cytomegalovirus (CMV) immediate early 

                     promoter"

     misc_feature    591..671

                     /note="MCS"

                     /note="multiple cloning site"

     regulatory      673..682

                     /regulatory_class="other"

                     /note="Kozak sequence"

                     /note="vertebrate consensus sequence for strong initiation 

                     of translation (Kozak, 1987)"

     CDS             679..1398

                     /codon_start=1

                     /product="cyan variant of GFP"

                     /note="CFP"

                     /note="mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK

                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     polyA_signal    1521..1642

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1649..2104)

                     /direction=LEFT

                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        2131..2235

                     /gene="bla"

                     /note="AmpR promoter"

     promoter        2237..2594

                     /note="SV40 promoter"

                     /note="SV40 enhancer and early promoter"

     rep_origin      2445..2580

                     /note="SV40 ori"

                     /note="SV40 origin of replication"

     CDS             2629..3423

                     /codon_start=1

                     /gene="aph(3')-II (or nptII)"

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /note="NeoR/KanR"

                     /note="confers resistance to neomycin, kanamycin, and G418 

                     (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3655..3702

                     /note="HSV TK poly(A) signal"

                     /note="herpes simplex virus thymidine kinase 

                     polyadenylation signal (Cole and Stacy, 1985)"

     rep_origin      4031..4619

                     /direction=RIGHT

                     /note="ori"

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 

                     replication"

ORIGIN

        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg

       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt

      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca

      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc

      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta

      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac

      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg

      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg

      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt

      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta

      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc

      721 atcctggtcg agctggacgg cgacgtaaac ggccacaggt tcagcgtgtc cggcgagggc

      781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg

      841 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcagccgc

      901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc

      961 caggagcgta ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag

     1021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac

     1081 ggcaacatcc tggggcacaa gctggagtac aactacatca gccacaacgt ctatatcacc

     1141 gccgacaagc agaagaacgg catcaaggcc cacttcaaga tccgccacaa catcgaggac

     1201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg

     1261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag

     1321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg

     1381 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg

     1441 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa

     1501 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca

     1561 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt

     1621 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc

     1681 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc

     1741 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag

     1801 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc

     1861 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa

     1921 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg

     1981 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt

     2041 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc

     2101 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa

     2161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat

     2221 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt

     2281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt

     2341 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc

     2401 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc

     2461 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg

     2521 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc

     2581 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa

     2641 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg

     2701 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc

     2761 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca

     2821 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc

     2881 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca

     2941 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat

     3001 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca

     3061 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg

     3121 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc

     3181 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct

     3241 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct

     3301 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac

     3361 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc

     3421 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag

     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg

     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg

     3601 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa

     3661 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt

     3721 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg

     3781 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca

     3841 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt

     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga

     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca

     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac

     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg

     4141 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag

     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac

     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt

     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg

     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc

     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc

     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc

     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa

     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt

     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat

//

 

Caution:
Product is for research use only!

 

 

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