LOCUS       sequence-423504-        5709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mTagBFP2-FRB-Rab7 (late endosome marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5709)
  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 5709)
  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-Rab7
FEATURES             Location/Qualifiers
     source          1..5709
                     /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    2497..2618
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2534..2553)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2588..2607
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2625..3080)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2762..2783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2974..2993
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3107..3211
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3213..3570
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3213..3404
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3236..3256)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3421..3556
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3605..4399
                     /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             3605..4399
                     /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(3659..3678)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4269..4288
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4587..4606)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4631..4678
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5007..5595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5496..5515
                     /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 aagcttcgat gacctctagg
     1741 aagaaagtgt tgctgaaggt tatcatcctg ggagattctg gagttggtaa gacatcactc
     1801 atgaaccagt atgtgaacaa gaaattcagt aatcagtaca aagctacaat aggagcagac
     1861 tttctgacaa aggaggtgat ggtggatgac agactagtta caatgcagat ctgggacaca
     1921 gcaggccagg aacggttcca gtcccttggt gtggccttct acagaggtgc agactgctgc
     1981 gttctggtat ttgacgttac tgcccccaac acattcaaaa ccctcgatag ctggagagat
     2041 gagtttctca tccaggccag tccccgggat cctgaaaact tccctttcgt tgtcctagga
     2101 aacaagattg acctcgaaaa cagacaagtg gccacaaagc gggcacaggc ctggtgctac
     2161 agcaaaaaca acattcccta cttcgagacc agtgccaagg aggccatcaa tgtggagcag
     2221 gcgttccaga cgattgcaag gaatgcactt aaacaggaaa cagaggtgga gctgtacaat
     2281 gaattccctg aacccatcaa actggacaag aacgaccggg ccaagacctc agcggaaagc
     2341 tgcagttgct gaggtaccgc gggcccggga tccaccggat ctagataact gatcataatc
     2401 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     2461 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2521 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2581 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     2641 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2701 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2761 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2821 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2881 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2941 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3001 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3061 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3121 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3181 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     3241 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3301 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3361 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3421 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3481 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3541 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     3601 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3661 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3721 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3781 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3841 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3901 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3961 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4021 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4081 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     4141 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4201 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4261 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4321 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4381 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4441 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4501 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4561 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4621 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     4681 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     4741 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     4801 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     4861 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4921 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4981 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5041 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5101 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5161 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5221 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5281 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5341 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5401 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5461 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5521 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5581 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5641 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5701 gccatgcat
//
