LOCUS       sequence-247521-        4923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a chimera of the mitochondria-targeting signal of TOM20, 
            SBP tag and GFP (fluorescent tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4923)
  AUTHORS   Guardia CM, De Pace R, Sen A, Saric A, Jarnik M, Kolin DA, Kunwar A,
            Bonifacino JS
  TITLE     Reversible association with motor proteins (RAMP): A 
            streptavidin-based method to manipulate organelle positioning.
  JOURNAL   PLoS Biol. 2019 May 17;17(5):e3000279. doi: 
            10.1371/journal.pbio.3000279. eCollection 2019 May.
  PUBMED    31100061
REFERENCE   2  (bases 1 to 4923)
  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: "2019-05-17"; volume: "17(5):e3000279. doi"; pages: " 
            10.1371/journal.pbio.3000279. eCollection 2019 May"
COMMENT     Sequence Label: TOM20*-SBP-GFP
FEATURES             Location/Qualifiers
     source          1..4923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     296..315
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(614..633)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    658..705
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1034..1622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1523..1542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1797..2100
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2101..2304
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2255..2275
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2449..2559
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     regulatory      2599..2608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2605..2607,2608..2610,2611..3324)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2650..2671)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2911..2930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3258..3279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3447..3568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3484..3503)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3538..3557
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3575..4030)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3712..3733)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3924..3943
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4057..4161
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4163..4520
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4163..4354
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4186..4206)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4371..4506
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4555..4923,1..426)
                     /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             join(4555..4923,1..426)
                     /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(4609..4628)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
       61 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
      121 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
      181 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
      241 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
      301 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
      361 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
      421 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
      481 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
      541 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
      601 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
      661 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
      721 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
      781 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
      841 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
      901 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
      961 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1021 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1081 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1141 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     1201 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1261 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1321 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1381 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1441 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1501 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1561 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     1621 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     1681 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt
     1741 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     1801 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     1861 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     1921 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1981 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     2041 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     2101 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     2161 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     2221 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     2281 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
     2341 actcagatct cgagctcaat ggtgggtcgg aacagcgcca tcgccgccgg tgtatgcggg
     2401 gcccttttca ttgggtactg catctacttc gaccgcaaaa gacgaagtga cgagaagacc
     2461 actggttggc gaggtggaca cgttgttgaa ggactggctg gggaacttga acaacttcgt
     2521 gcacgactgg agcatcaccc acaaggtcaa cgtgaaccac tgcagtcgac ggtaccgcgg
     2581 gcccgggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     2641 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2701 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2761 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2821 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2881 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2941 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3001 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3061 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3121 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3181 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc
     3241 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3301 ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca
     3361 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3421 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3481 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3541 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta
     3601 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3661 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     3721 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3781 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     3841 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3901 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     3961 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4021 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4081 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4141 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4201 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4261 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4321 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4381 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4441 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4501 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4561 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4621 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4681 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     4741 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4801 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4861 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4921 ctg
//
