LOCUS       sequence-423509-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mTagBFP2-FRB-Rab5 (early endosome marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  AUTHORS   Ajiki M, Yoshikawa M, Miyazaki T, Kawasaki A, Aoki K, Nakatsu F, 
            Tsukiji S
  TITLE     ORP9-PH domain-based fluorescent reporters for visualizing 
            phosphatidylinositol 4-phosphate dynamics in living cells.
  JOURNAL   RSC Chem Biol. 2024 Apr 24;5(6):544-555. doi: 10.1039/d3cb00232b. 
            eCollection 2024 Jun 5.
  PUBMED    38846081
REFERENCE   2  (bases 1 to 5739)
  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: 
            "10.1039/d3cb00232b"; journalName: "RSC Chem Biol"; date: 
            "2024-04-24- 24"; volume: "5"; issue: "6"; pages: "544-555"
COMMENT     Sequence Label: pCMV-mTagBFP2-FRB-Rab5
FEATURES             Location/Qualifiers
     source          1..5739
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1323
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             1417..1695
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     polyA_signal    2527..2648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2564..2583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2618..2637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2655..3110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2792..2813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3004..3023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3137..3241
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3243..3600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3243..3434
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3266..3286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3451..3586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3635..4429
                     /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             3635..4429
                     /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(3689..3708)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4299..4318
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4617..4636)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4661..4708
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5037..5625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5526..5545
                     /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 ccggtcgcca ccatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag
      661 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
      721 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
      781 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
      841 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
      901 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
      961 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     1021 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     1081 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     1141 aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     1201 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     1261 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt
     1321 aattccggac tcagatctgg tggcggaggc tcgggcggag gtgggtcggg tggcggcgga
     1381 tctcgagctg gaggtgctgg tgctatccta tctagaatcc tctggcatga gatgtggcat
     1441 gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt
     1501 gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct gaaggaaaca
     1561 tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac
     1621 atgaaatcag ggaatgtcaa ggacctcacc caagcctggg acctctatta tcatgtgttc
     1681 cgacgaatct caaagggtgg tagtgctggt ggtagtgctc aagcttcgaa ttctgcagtc
     1741 gacggtacca tggctaatcg aggagcaaca agacccaacg ggccaaatac tggaaataaa
     1801 atatgccagt tcaaactagt acttctggga gagtctgctg ttggcaaatc aagcctagtg
     1861 cttcgttttg tgaagggcca atttcatgaa tttcaagaga gtaccatagg ggctgctttt
     1921 ctaacccaaa ctgtgtgtct tgatgataca acagtaaagt ttgaaatatg ggatacagct
     1981 ggtcaagaac gataccatag cttagcacca atgtactaca gaggagcaca agcagccata
     2041 gttgtatatg atatcacaaa tgaggagtcc tttgccagag ccaaaaactg ggttaaagaa
     2101 cttcagaggc aagccagtcc taacattgta atagctttat caggaaacaa ggctgatctt
     2161 gcaaataaaa gagctgtcga tttccaggaa gcacagtcct atgcagatga caacagttta
     2221 ttattcatgg agacatcagc taaaacatcg atgaatgtaa atgaaatatt catggcaata
     2281 gctaaaaagt tgccaaagaa cgaaccacag aatccaggag caaattctgc cagaggaaga
     2341 ggagtagacc ttactgaacc cacgcagcca accaggagtc agtgttgtag taactaagga
     2401 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2461 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2521 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2581 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2641 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2701 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2761 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2821 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2881 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2941 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3001 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3061 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     3121 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3181 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3241 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3301 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3361 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3421 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3481 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3541 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3601 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3661 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3721 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3781 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3841 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3901 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3961 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4021 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4081 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4141 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4201 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4261 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4321 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4381 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4441 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4501 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4561 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4621 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4681 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4741 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4801 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4861 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4921 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4981 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5041 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5101 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5161 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5221 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5281 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5341 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5401 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5461 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5521 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5581 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5641 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5701 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
