LOCUS       sequence-254957-        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  JOURNAL   Unpublished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            "Unpublished"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        27..330
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        331..534
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     485..505
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             593..667
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      686..695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             692..1066
                     /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             1073..1102
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1239..1360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1276..1295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1330..1349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1367..1822)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1504..1525)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1716..1735
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1849..1953
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1955..2312
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1955..2146
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1978..1998)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2163..2298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2347..3141
                     /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             2347..3141
                     /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(2401..2420)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3011..3030
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3329..3348)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3373..3420
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3749..4337
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4238..4257
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
       61 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      121 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      181 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      241 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      301 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      361 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      421 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      481 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
      541 gtgaaccgtc agatccgcta gcgctaccgg actcagatcc gaattcgcca ccatgtccgt
      601 cctgacgccg ctgctgctgc ggggcttgac aggctcggcc cggcggctcc cagtgccgcg
      661 cgccaagatc cattcgttga gatctgccac catggagcat gttgcctttg gcagtgagga
      721 catcgagaac actctggcca aaatggacga cggacaactg gatgggttgg cctttggcgc
      781 aattcagctc gatggtgacg ggaatatcct gcagtacaat gctgctgaag gagacatcac
      841 aggcagagat cccaaacagg tgattgggaa gaacttcttc aaggatgttg cacctggaac
      901 ggattctccc gagttttacg gcaaattcaa ggaaggcgta gcgtcaggga atctgaacac
      961 catgttcgaa tggatgatac cgacaagcag gggaccaacc aaggtcaaga tacacatgaa
     1021 gaaagccctt tccggtgaca gctattgggt ctttgtgaaa cgggtgggat ccgaacaaaa
     1081 gcttatttct gaagaggact tgtaatagcc cgggtaagcg gccgcgactc tagatcataa
     1141 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     1201 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     1261 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     1321 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     1381 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1441 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1501 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1561 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     1621 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     1681 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     1741 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     1801 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1921 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     1981 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2041 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2101 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2161 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2221 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     2281 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     2341 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     2401 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     2461 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     2521 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     2581 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     2641 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     2701 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     2761 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     2821 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     2881 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     2941 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3001 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3061 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3121 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     3181 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3241 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3301 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     3361 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     3421 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     3481 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     3541 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     3601 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3661 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3721 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3781 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3841 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3901 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3961 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4021 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4081 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4141 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4201 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4261 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4321 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4381 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4441 ccgccatgca ttagttatta atagtaatca attacggggt catta
//
