LOCUS       sequence-344825-        6805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the peroxisomal variant (splice variant 4) of Miro with an
            N-terminal EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6805)
  AUTHORS   Guillen-Samander A, Leonzino M, Hanna MG, Tang N, Shen H, De Camilli
            P
  TITLE     VPS13D bridges the ER to mitochondria and peroxisomes via Miro.
  JOURNAL   J Cell Biol. 2021 May 3;220(5). pii: 212021. doi: 
            10.1083/jcb.202010004.
  PUBMED    33891013
REFERENCE   2  (bases 1 to 6805)
  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: "J Cell 
            Biol."; date: "2021-05-3"; volume: "220(5). pii: 212021. doi"; 
            pages: " 10.1083/jcb.202010004"
COMMENT     Sequence Label: EGFP-Miro1v4
FEATURES             Location/Qualifiers
     source          1..6805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(249..251,252..254,255..965)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(249..251,252..254,255..965)
                     /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(294..315)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(555..574)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     902..923
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3229..3350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3266..3285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3320..3339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3357..3812)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3494..3515)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3706..3725
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3839..3943
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3945..4302
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3945..4136
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3968..3988)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4153..4288
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4337..5131
                     /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             4337..5131
                     /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(4391..4410)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5001..5020
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5319..5338)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5363..5410
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5739..6327
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6228..6247
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6502..6805
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
      601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
      661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
      721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
      781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
      901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      961 acaagtccgg actcagatct cgagtgggat ctatgaagaa agacgtgcgg atcctgctgg
     1021 tgggagaacc tagagttggg aagacatcac tgattatgtc tctggtcagt gaagaatttc
     1081 cagaagaggt tcctccccgg gcagaagaaa tcaccattcc agctgatgtc accccagaga
     1141 gagttccaac acacattgta gattactcag aagcagaaca gagtgatgaa caacttcatc
     1201 aagaaatatc tcaggctaat gtcatctgta tagtgtatgc cgttaacaac aagcattcta
     1261 ttgataaggt aacaagtcga tggattcctc tcataaatga aagaacagac aaagacagca
     1321 ggctgccttt aatattggtt gggaacaaat ctgatctggt ggaatatagt agtatggaga
     1381 ccatccttcc tattatgaac cagtatacag aaatagaaac ctgtgtggag tgttcagcga
     1441 aaaacctgaa gaacatatca gagctctttt attacgcaca gaaagctgtt cttcatccta
     1501 cagggcccct gtactgccca gaggagaagg agatgaaacc agcttgtata aaagccctta
     1561 ctcgtatatt taaaatatct gatcaagata atgatggtac tctcaatgat gctgaactca
     1621 acttctttca gaggatttgt ttcaacactc cattagctcc tcaagctctg gaggatgtca
     1681 agaatgtagt cagaaaacat ataagtgatg gtgtggctga cagtgggttg accctgaaag
     1741 gttttctctt tttacacaca ctttttatcc agagagggag acacgaaact acttggactg
     1801 tgcttcgacg atttggttat gatgatgacc tggatttgac acctgaatat ttgttccccc
     1861 tgctgaaaat acctcctgat tgcactactg aattaaatca tcatgcatat ttatttctcc
     1921 aaagcacctt tgacaagcat gatttggata gagactgtgc tttgtcacct gatgagctta
     1981 aagatttatt taaagttttc ccttacatac cttgggggcc agatgtgaat aacacagttt
     2041 gtaccaatga aagaggctgg ataacctacc agggattcct ttcccagtgg acgctcacga
     2101 cttatttaga tgtacagcgg tgcctggaat atttgggcta tctaggctat tcaatattga
     2161 ctgagcaaga gtctcaagct tcagctgtta cagtgacaag agataaaaag atagacctgc
     2221 agaaaaaaca aactcaaaga aatgtgttca gatgtaatgt aattggagtg aaaaactgtg
     2281 ggaaaagtgg agttcttcag gctcttcttg gaagaaactt aatgaggcag aagaaaattc
     2341 gtgaagatca taaatcctac tatgcgatta acactgttta tgtatatgga caagagaaat
     2401 acttgttgtt gcatgatatc tcagaatcgg aatttctaac tgaagctgaa atcatttgtg
     2461 atgttgtatg cctggtatat gatgtcagca atcccaaatc ctttgaatac tgtgccagga
     2521 tttttaagca acactttatg gacagcagaa taccttgctt aatcgtagct gcaaagtcag
     2581 acctgcatga agttaaacaa gaatacagta tttcacctac tgatttctgc aggaaacaca
     2641 aaatgcctcc accacaagcc ttcacttgca atactgctga tgcccccagt aaggatatct
     2701 ttgttaaatt gacaacaatg gccatgtatc cagaggatca ttacagagac agactctccc
     2761 gagacatggg ccacactgat agaatagaga atttgagaaa aatctgggtc tttctaaaaa
     2821 ctgctttcca tgcccggtta cgctgtatgt gcacctgcaa caggtgtaca ttttgcatct
     2881 gtcagaactt cctcaactca gacttgctgc aatctgtaaa gaacaaaatc ttcactgcag
     2941 ttcttaacag gcacgtgaca caagctgacc tcaagagctc cacgttttgg cttcgagcaa
     3001 gttttggtgc tactgttttt gcagttttgg gctttgctat gtacaaagca ttattgaaac
     3061 agcgatgaga attctgcagt cgacggtacc gcgggcccgg gatccaccgg atctagataa
     3121 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3181 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3241 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3301 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     3361 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3421 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3481 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3541 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3601 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3661 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3721 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3781 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     3841 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3901 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     3961 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4021 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4081 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4141 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4201 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4261 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4321 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4381 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4441 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4501 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4561 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4621 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4681 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4741 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4801 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4861 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4921 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4981 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5041 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5101 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5161 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5221 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5281 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5341 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5401 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5461 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5521 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5581 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5641 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5701 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5761 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5821 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5881 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5941 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6001 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6061 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6121 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6181 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6241 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6301 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6361 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6421 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
     6481 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     6541 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     6601 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     6661 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     6721 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6781 cgtattagtc atcgctatta ccatg
//
