pEYFP-Actin细胞骨架定位质粒

基本信息

启动子:	CMV
复制子:	pUC ori,f1 ori
终止子:	SV40 poly(A) signal
质粒分类:	哺乳细胞，荧光蛋白报告载体
质粒大小:	5820bp
原核抗性:	Kan
筛选标记:	Neo
克隆菌株:	DH5α
培养条件:	37℃，有氧 LB
表达宿主:	哺乳细胞
诱导方式:	无须诱导，瞬时表达
5'测序引物:	CMV-F：CGCAAATGGGCGGTAGGCGTG
3'测序引物:	根据序列设计引物

质粒简介

    pEYFP-Actin encodes a fusion of the enhanced yellow fluorescent protein (EYFP) and human cytoplasmic β-actin. EYFP is an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein (GFP). The EYFP gene contains the four amino acid substitutions previously published as GFP-10C: Ser-65 to Gly; Val-68 to Leu; Ser-72 to Ala; and Thr-203 to Tyr. It also contains the Phe-64 to Leu mutation of GFPmut1. The fluorescence excitation maximum of EYFP is 513 nm; the emission spectrum has a peak at 527 nm (in the yellow-green region). When excited at 513 nm, the Em of EYFP is 36,500 cm–1M–1 and the fluorescence quantum yield is 0.63, resulting in a bright fluorescent signal. The fluorescence observed is roughly equivalent to that from EGFP

质粒图谱

质粒序列

LOCUS       Exported                5820 bp ds-DNA    circular SYN 19-10-2015
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5820)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-10-19   from MLCC
FEATURES             Location/Qualifiers
     source          1..5820
                     /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"
     CDS             613..1329
                     /codon_start=1
                     /product="enhanced YFP"
                     /note="EYFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    2608..2729
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2736..3191)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3218..3322
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        3324..3681
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      3532..3667
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             3716..4510
                     /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    4742..4789
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      5118..5706
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct atggatgatg atatcgccgc gctcgtcgtc
     1381 gacaacggct ccggcatgtg caaggccggc ttcgcgggcg acgatgcccc ccgggccgtc
     1441 ttcccctcca tcgtggggcg ccccaggcac cagggcgtga tggtgggcat gggtcagaag
     1501 gattcctatg tgggcgacga ggcccagagc aagagaggca tcctcaccct gaagtacccc
     1561 atcgagcacg gcatcgtcac caactgggac gacatggaga aaatctggca ccacaccttc
     1621 tacaatgagc tgcgtgtggc tcccgaggag caccccgtgc tgctgaccga ggcccccctg
     1681 aaccccaagg ccaaccgcga gaagatgacc cagatcatgt ttgagacctt caacacccca
     1741 gccatgtacg ttgctatcca ggctgtgcta tccctgtacg cctctggccg taccactggc
     1801 atcgtgatgg actccggtga cggggtcacc cacactgtgc ccatctacga ggggtatgcc
     1861 ctcccccatg ccatcctgcg tctggacctg gctggccggg acctgactga ctacctcatg
     1921 aagatcctca ccgagcgcgg ctacagcttc accaccacgg ccgagcggga aatcgtgcgt
     1981 gacattaagg agaagctgtg ctacgtcgcc ctggacttcg agcaagagat ggccacggct
     2041 gcttccagct cctccctgga gaagagctac gagctgcctg acggccaggt catcaccatt
     2101 ggcaatgagc ggttccgctg ccctgaggca ctcttccagc cttccttcct gggcatggag
     2161 tcctgtggca tccacgaaac taccttcaac tccatcatga agtgtgacgt ggacatccgc
     2221 aaagacctgt acgccaacac agtgctgtct ggcggcacca ccatgtaccc tggcattgcc
     2281 gacaggatgc agaaggagat cactgccctg gcacccagca caatgaagat caagatcatt
     2341 gctcctcctg agcgcaagta ctccgtgtgg atcggcggct ccatcctggc ctcgctgtcc
     2401 accttccagc agatgtggat cagcaagcag gagtatgacg agtccggccc ctccatcgtc
     2461 caccgcaaat gcttctaggg atccaccgga tctagataac tgatcataat cagccatacc
     2521 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2581 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2641 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2701 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     2761 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2821 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2881 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2941 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3001 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3061 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3121 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3181 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3301 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3361 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3421 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3481 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3541 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3601 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3661 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3721 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3781 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3841 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     3901 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3961 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4021 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4081 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4141 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4201 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4261 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4321 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4381 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4441 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4501 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4561 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4621 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4681 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4741 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     4801 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     4861 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     4921 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     4981 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5041 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5101 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5161 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5221 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5281 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5341 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5401 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5461 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5521 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5581 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5641 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5701 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5761 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat