LOCUS       sequence-354822-        6154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP with microtubule-targeting sequence in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6154)
  AUTHORS   Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, 
            Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y
  TITLE     Folding Latency of Fluorescent Proteins Affects the Mitochondrial 
            Localization of Fusion Proteins.
  JOURNAL   Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi: 
            10.1247/csf.19028. Epub 2019 Nov 15.
  PUBMED    31735740
REFERENCE   2  (bases 1 to 6154)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1247/csf.19028";
            journalName: "Cell Struct Funct"; date: "2019-12-26- 26"; volume: 
            "44"; issue: "2"; pages: "183-194"
COMMENT     Sequence Label: pFX-beta-tubulin-EGFP
FEATURES             Location/Qualifiers
     source          1..6154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1998..2000,2001..2003,2004..2714)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1998..2000,2001..2003,2004..2714)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2043..2064)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2304..2323)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2651..2672
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2942..3063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2979..2998)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3033..3052
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3070..3525)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3207..3228)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3419..3438
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3552..3656
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3658..4015
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3658..3849
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3681..3701)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3866..4001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4050..4844
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4050..4844
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4104..4123)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4714..4733
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5032..5051)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5076..5123
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5452..6040
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5941..5960
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 ccggttctag agccatgctc gagaccacca tgagggaaat cgtgcacttg caggccgggc
      661 agtgcggcaa ccaaatcggc gccaagtttt gggaggtgat cagcgatgag cacggcatcg
      721 accccacggg cacctaccac ggggacagcg acctgcagct ggaacgcatc aacgtgtact
      781 acaatgaggc caccggcggc aagtacgtgc cccgcgccgt gctcgtggat ctggagcccg
      841 gcaccatgga ctccgtgcgc tcggggccct tcgggcagat cttccggccg gacaacttcg
      901 ttttcggtca gagtggtgct gggaacaact gggccaaggg gcactacaca gaaggcgcgg
      961 agctggtgga ctcggtgctg gatgttgtga gaaaggaggc tgagagctgt gactgcctgc
     1021 agggtttcca gctgacccac tccctgggtg gggggactgg gtctgggatg ggtaccctcc
     1081 tcatcagcaa gatccgggag gagtacccag acaggatcat gaacacgttt agtgtggtgc
     1141 cttcgcccaa agtgtcagac acagtggtgg agccctacaa cgccaccctc tcagtccacc
     1201 agctcgtaga aaacacagac gagacctact gcattgataa cgaagctctc tacgacattt
     1261 gcttcagaac cctaaagctg accacgccca cctatggtga cctgaaccac ctggtgtctg
     1321 ctaccatgag tggggtcacc acctgcctgc gcttcccagg ccagctcaat gctgacctgc
     1381 ggaagctggc tgtgaacatg gtcccgtttc cccggctgca cttcttcatg cccggctttg
     1441 ccccactgac cagccggggc agccagcagt accgggcgct gaccgtgccc gagctcaccc
     1501 agcagatgtt tgatgccaag aacatgatgg ctgcctgcga cccccgccat ggccgctacc
     1561 tgacggttgc cgccgtgttc aggggccgca tgtccatgaa ggaggtggat gagcaaatgc
     1621 ttaatgtcca aaacaaaaac agcagctatt ttgttgagtg gatccccaac aatgtgaaaa
     1681 cggctgtctg tgacatccca cctcgggggc taaaaatgtc cgccaccttc attggcaaca
     1741 gcacggccat ccaggagctg ttcaagcgca tctccgagca gttcacggcc atgttccggc
     1801 gcaaggcctt cctgcactgg tacacgggcg agggcatgga cgagatggag ttcaccgagg
     1861 ccgagagcaa catgaatgac ctggtgtccg agtaccagca gtaccaggat gccacagccg
     1921 aggaggaggg cgagttcgag gaggaggctg aggaggaggt ggcctgcggc cgcggaggat
     1981 ccggaggaga attcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2041 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2101 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2161 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2221 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2281 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2341 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2401 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2461 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2521 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2581 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2641 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2701 acgagctgta caagagatct ctgactgact gagatgcgcg gatgtcgaac ggcagttcgg
     2761 ccgaataggc cgacagccgg ccgtcggccg acgacacggc cagggccggc ggggatccac
     2821 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2881 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2941 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3001 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3061 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3121 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3181 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3241 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3301 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3361 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3421 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3481 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3541 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3601 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     3661 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3721 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3781 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3841 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3901 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3961 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     4021 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4081 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4141 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4201 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     4261 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4321 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4381 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4441 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4501 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4561 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4621 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4681 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4741 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4801 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4861 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4921 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4981 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     5041 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5101 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     5161 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     5221 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     5281 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     5341 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5401 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5461 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5521 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5581 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5641 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5701 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5761 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5821 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5881 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5941 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6001 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6061 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6121 tgattctgtg gataaccgta ttaccgccat gcat
//
