LOCUS       sequence-250357-        4485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mito-iFAST in mammalian cells (mitochondria labeling).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4485)
  TITLE     iFAST-containing plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4485)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAG497 mito-iFAST
FEATURES             Location/Qualifiers
     source          1..4485
                     /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"
     CDS             627..701
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      720..729
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             726..1100
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKIHMKKALSGDSYWVFVKRV"
     CDS             1107..1136
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1273..1394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1310..1329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1364..1383
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1401..1856)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1538..1559)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1750..1769
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1883..1987
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1989..2346
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1989..2180
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2012..2032)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2197..2332
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2381..3175
                     /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             2381..3175
                     /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(2435..2454)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3045..3064
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3363..3382)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3407..3454
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3783..4371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4272..4291
                     /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 ccggactcag atccgaattc gccaccatgt ccgtcctgac gccgctgctg ctgcggggct
      661 tgacaggctc ggcccggcgg ctcccagtgc cgcgcgccaa gatccattcg ttgagatctg
      721 ccaccatgga gcatgttgcc tttggcagtg aggacatcga gaacactctg gccaaaatgg
      781 acgacggaca actggatggg ttggcctttg gcgcaattca gctcgatggt gacgggaata
      841 tcctgcagta caatgctgct gaaggagaca tcacaggcag agatcccaaa caggtgattg
      901 ggaagaactt cttcaaggat gttgcacctg gaacggattc tcccgagttt tacggcaaat
      961 tcaaggaagg cgtagcgtca gggaatctga acaccatgtt cgaatggatg ataccgacaa
     1021 gcaggggacc aaccaaggtc aagatacaca tgaagaaagc cctttccggt gacagctatt
     1081 gggtctttgt gaaacgggtg ggatccgaac aaaagcttat ttctgaagag gacttgtaat
     1141 agcccgggta agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     1201 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     1261 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1321 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1381 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1441 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1501 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1561 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1621 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     1681 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     1741 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     1801 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     1861 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1921 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1981 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2041 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2101 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2161 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2221 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2281 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2341 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2401 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2461 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2521 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     2581 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     2641 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     2701 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     2761 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     2821 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     2881 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     2941 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3001 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3061 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3121 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3181 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3241 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3301 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     3361 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     3421 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     3481 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     3541 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     3601 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     3661 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3721 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3781 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3841 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3901 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3961 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4021 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4081 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4141 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4201 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4261 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4321 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4381 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4441 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
