LOCUS       sequence-354827-        5379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP with early endosome-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5379)
  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 5379)
  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-EGFP-Rab5
FEATURES             Location/Qualifiers
     source          1..5379
                     /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(627..629,630..632,633..1343)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(627..629,630..632,633..1343)
                     /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(672..693)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(933..952)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1280..1301
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2167..2288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2204..2223)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2258..2277
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2295..2750)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2432..2453)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2644..2663
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2777..2881
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2883..3240
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2883..3074
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2906..2926)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3091..3226
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3275..4069
                     /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             3275..4069
                     /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(3329..3348)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3939..3958
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4257..4276)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4301..4348
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4677..5265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5166..5185
                     /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 aagtactgaa ttcaccatgg tgagcaaggg cgaggagctg ttcaccgggg
      661 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      721 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      781 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      841 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      901 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      961 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1021 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1081 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1141 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1201 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1261 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1321 tcggcatgga cgagctgtac aagagatctg gaggatccgg aggactcgag atggctaatc
     1381 gaggagcaac aagacccaac gggccaaata ctggaaataa aatatgccag ttcaaactgg
     1441 tccttctagg agagtctgct gttggcaaat caagcctggt tcttcgcttt gtgaaaggcc
     1501 aatttcatga atttcaagag agtaccattg gggctgcctt tctaacccaa actgtgtgtc
     1561 ttgatgacac aacagtaaaa tttgaaatat gggatacagc tggtcaagaa cggtatcata
     1621 gcttagcacc aatgtactac cgaggagcac aagcagccat agttgtgtat gatatcacaa
     1681 atgaggaatc ctttgcgaga gcaaaaaact gggttaaaga acttcaaagg caagcaagtc
     1741 ctaatattgt gatagctttg tcaggaaaca aagctgactt agcaaataaa agagctgttg
     1801 acttccagga agcacagtcc tatgcagatg acaacagctt attatttatg gagacatcag
     1861 ctaagacatc aatgaatgta aatgaaatat ttatggcaat agctaaaaag ctgccaaaga
     1921 atgaaccaca gaatcctggt gcaaactcag ccagaggacg aggagtagac cttactgagc
     1981 ctgcacagcc agccagaagc cagtgttgta gtaactgagc ggccgctgac tgactgaaga
     2041 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2101 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2161 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2221 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2281 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2341 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2401 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2461 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2521 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2581 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2641 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2701 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     2761 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2821 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2881 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2941 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3001 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3061 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3121 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3181 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3241 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3301 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3361 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3421 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3481 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3541 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3601 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3661 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3721 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3781 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3841 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3901 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3961 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4021 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4081 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4141 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4201 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4261 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4321 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4381 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4441 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4501 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4561 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4621 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4681 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4741 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4801 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4861 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4921 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4981 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5041 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5101 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5161 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5221 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5281 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5341 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
