LOCUS       sequence-435222-        4587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a mitochondrially-targeted SNAP tag for mammalian cell 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4587)
  AUTHORS   Harding O, Holzer E, Riley JF, Martens S, Holzbaur ELF
  TITLE     Damaged mitochondria recruit the effector NEMO to activate NF-kappaB
            signaling.
  JOURNAL   Mol Cell. 2023 Sep 7;83(17):3188-3204.e7. doi: 
            10.1016/j.molcel.2023.08.005.
  PUBMED    37683611
REFERENCE   2  (bases 1 to 4587)
  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"; doi: 
            "10.1016/j.molcel.2023.08"; journalName: "Mol Cell"; date: 
            "2023-09-7- 7"; volume: "83"; issue: "17"; pages: "3188-3204.e7"
COMMENT     Sequence Label: Mito-SNAP
FEATURES             Location/Qualifiers
     source          1..4587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..367
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        368..571
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     522..542
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             600..674
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      702..711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             708..1253
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     polyA_signal    1378..1499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1415..1434)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1469..1488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1506..1961)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1643..1664)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1855..1874
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1988..2092
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2094..2451
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2094..2285
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2117..2137)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2302..2437
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2486..3280
                     /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             2486..3280
                     /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(2540..2559)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3150..3169
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3468..3487)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3512..3559
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3888..4476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4377..4396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
       61 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      121 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      181 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      241 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      301 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      361 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      421 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      481 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      541 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagca
      601 tgtccgtcct gacgccgctg ctgctgcggg gcttgacagg ctcggcccgg cggctcccag
      661 tgccgcgcgc caagatccat tcgttggggg atccaccggt cgccaccatg gacaaagact
      721 gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg tctgggtgcg
      781 aacagggcct gcaccgtatc atcttcctgg gcaaaggaac atctgccgcc gacgccgtgg
      841 aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag gccaccgcct
      901 ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg ccagccctgc
      961 accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa ctgctgaaag
     1021 tggtgaagtt cggagaggtc atcagctaca gccacctggc cgccctggcc ggcaatcccg
     1081 ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt ctgatcccct
     1141 gccaccgggt ggtgcagggc gacctggacg tggggggcta cgagggcggg ctcgccgtga
     1201 aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg ggttgagcgg
     1261 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     1321 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     1381 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1441 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     1501 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     1561 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     1621 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     1681 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     1741 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     1801 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     1861 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     1921 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     1981 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2041 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     2101 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2161 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2221 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2281 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2341 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     2401 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     2461 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     2521 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     2581 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     2641 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     2701 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     2761 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     2821 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     2881 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     2941 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3001 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3061 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3121 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3181 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3241 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3301 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3361 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3421 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     3481 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     3541 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     3601 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     3661 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     3721 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3781 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4021 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4561 tctgtggata accgtattac cgccatg
//
