LOCUS       sequence-453949-        7435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-Rab5 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7435)
  AUTHORS   Kurikawa Y, Koyama-Honda I, Tamura N, Koike S, Mizushima N
  TITLE     Organelle landscape analysis using a multiparametric particle-based 
            method.
  JOURNAL   PLoS Biol. 2024 Sep 17;22(9):e3002777. doi: 
            10.1371/journal.pbio.3002777. eCollection 2024 Sep.
  PUBMED    39288101
REFERENCE   2  (bases 1 to 7435)
  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"; journalName: "PLoS 
            Biol."; date: "2024-09-17"; volume: "22(9):e3002777. doi"; pages: " 
            10.1371/journal.pbio.3002777. eCollection 2024 Sep"
COMMENT     Sequence Label: pMRX-IPU-GFP-Rab5
FEATURES             Location/Qualifiers
     source          1..7435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    739..1096
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1545..1561
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1587..1961
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1899..1915
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1994..1996,1997..1999,2000..2710)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1994..1996,1997..1999,2000..2710)
                     /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(2039..2060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2300..2319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2647..2668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3421..3987
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3437..3989
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3581..3598)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3808..3827
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4007..4606
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4007..4026)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4503..4523
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5360..5377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5531..6119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5611..5630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6283..7074,7075..7143))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6906..6925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7144..7248)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7316..7334
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg ctagagaacc atcagatgtt tccagggtgc cccaaggacc
      421 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
      481 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
      541 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
      601 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
      661 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
      721 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
      781 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
      841 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
      901 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
      961 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc
     1021 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     1081 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     1141 tgtctgaaaa tatgggcccg ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc
     1621 ttgtaaactt ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc
     1681 acttacaggc tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc
     1741 aagaacaact ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg
     1801 tccgccgaca ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc
     1861 tgaccacccc caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg
     1921 tgaaggctgc cgaccccggg ggtggaccat cctctagact gccggatctg ccatcgatat
     1981 aaagcttgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2041 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2101 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2161 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2221 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2281 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2341 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2401 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2461 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2521 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2581 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2641 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2701 gctgtacaag ggttcaggct cgggatccgg gaattcgggt ctcgagagcg gccgcatggc
     2761 taatcgagga gcaacaagac ccaacgggcc aaatactgga aataaaatat gccagttcaa
     2821 actggtcctt ctaggagagt ctgctgttgg caaatcaagc ctggttcttc gctttgtgaa
     2881 aggccaattt catgaatttc aagagagtac cattggggct gcctttctaa cccaaactgt
     2941 gtgtcttgat gacacaacag taaaatttga aatatgggat acagctggtc aagaacggta
     3001 tcatagctta gcaccaatgt actaccgagg agcacaagca gccatagttg tgtatgatat
     3061 cacaaatgag gaatcctttg cgagagcaaa aaactgggtt aaagaacttc aaaggcaagc
     3121 aagtcctaat attgtgatag ctttgtcagg aaacaaagct gacttagcaa ataaaagagc
     3181 tgttgacttc caggaagcac agtcctatgc agatgacaac agcttattat ttatggagac
     3241 atcagctaag acatcaatga atgtaaatga aatatttatg gcaatagcta aaaagctgcc
     3301 aaagaatgaa ccacagaatc ctggtgcaaa ctcagccaga ggacgaggag tagaccttac
     3361 tgagcctgca cagccagcca gaagccagtg ttgtagtaac tgagcggccg ctacgtaaat
     3421 tccgcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     3481 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     3541 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
     3601 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag tttcttgaag acaaacaacg
     3661 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     3721 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     3781 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     3841 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     3901 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     3961 gttttccttt gaaaaacacg atgataatat ggccacaacc gtcgacatga ccgagtacaa
     4021 gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc
     4081 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg
     4141 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     4201 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     4261 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     4321 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     4381 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     4441 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     4501 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     4561 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgagtcg accgcccgcc
     4621 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catggctcga cgataaaata
     4681 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4741 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     4801 gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc
     4861 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc
     4921 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa
     4981 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc
     5041 ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc
     5101 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga
     5161 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg
     5221 gggtctttca catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca
     5281 ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5341 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5401 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5461 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5521 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5581 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5641 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5701 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5761 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5821 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5881 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5941 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6001 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6061 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6121 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6181 ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg cggccggccg
     6241 caaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6301 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6361 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6421 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6481 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6541 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6601 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6661 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6721 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6781 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6841 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     6901 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6961 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7021 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7081 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7141 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7201 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7261 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     7321 gcgtatcacg aggccctttc gtcttcacta taggagcttg gcccattgca tacgttgtat
     7381 ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc atgtt
//
