LOCUS       sequence-419152-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mitochondrial localized ApaLI with E225A and K227A 
            mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  AUTHORS   Fu Y, Sacco O, DeBitetto E, Kanshin E, Ueberheide B, Sfeir A
  TITLE     Mitochondrial DNA breaks activate an integrated stress response to 
            reestablish homeostasis.
  JOURNAL   Mol Cell. 2023 Oct 8:S1097-2765(23)00753-0. doi: 
            10.1016/j.molcel.2023.09.026.
  PUBMED    37832546
REFERENCE   2  (bases 1 to 6705)
  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: "Mol Cell. 
            2023 Oct 8:S1097-2765(23)00753-0. doi: 
            10.1016/j.molcel.2023.09.026."
COMMENT     Sequence Label: pcDNA3-ApaLIE225A, K227A-HA
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(71..862,863..931))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGASKGGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     694..713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(932..1036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1111..1130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1302..1681
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1682..1885
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1836..1856
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1930..1949
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1930..1948
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2032..2106
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             3265..3291
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3325..3343)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3326..3343)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3363..3380)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3369..3593
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3639..4067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3726..3745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3936..3957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4076..4096)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4081..4410
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4261..4396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4323..4342
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4477..5271
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4531..4550)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5141..5160
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5445..5566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5482..5501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5536..5555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5615..5631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5615..5631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5628..5650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5639..5655
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5663..5669,5670..5687,5688..5693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5708..5729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5846..5863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6017..6605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6097..6116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
       61 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
      121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
      181 catctggccc cagtgctgca atgataccgc gagacccacc cttggaggct ccagatttat
      241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
      301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
      361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     1021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     1081 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1141 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     1201 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     1261 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     1321 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     1381 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     1441 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     1501 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1561 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1621 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1681 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     1741 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     1801 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     1861 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
     1921 tatcgaaatt aatacgactc actataggga gacccaagct agcttggtac cgagctcgga
     1981 tccactagta acggccgcca gtgtgctgga attcgggtac tccgtgccat catgtccgtc
     2041 ctgacgccgc tgctgctgcg gggcttgaca ggctcggccc ggcggctccc agtgccgcgc
     2101 gccaagatcc attcgttgcc gccggagggg aagcttatga ccacacgaca gcgcttatct
     2161 gctgagagat ctcagcagct tacccgtctg ctcactatca ctaaaaccgc taacatgcgg
     2221 gccttaatgg aggctagcga actcgctaaa gtaattgccc tggtggcggt tgacataggt
     2281 aagtcagacg agatggccag agctttcccc gtgctgtggc caaaaatcag tcctcagcaa
     2341 gaatattacg caacagcggt tgactggttc accaaccccg atgaaaccgt cacctctttc
     2401 gatgtggtgg atatgctaga tgccggcact tccctggacc aagacttcat gacttatctg
     2461 aaatgtctaa ctgaactgca caagcgacgg agaaaatacg gcctgatcct gcagaggcag
     2521 cctctaccaa cgatggtgca ggtttcgccc cgagcactga tggaatacgg acctgacttt
     2581 ccgcccgagg cactggctag ctggcttacc tggcggaagt ttttttacga ccttgacaac
     2641 aggtctgccc aggaaacagg gtacttgttc gaacccattc tggccgccgc catcggaggc
     2701 gaggcaaaat ctgccagaga gcgtgtcgta agacgcaccg acgatccaac gaagggcagg
     2761 caggtggatt gctggaaggt gctccctgat ggtacgcctc tggcttatgc gttggcgctt
     2821 cgggtgacaa ttgcagcgag cgggcaaggc agatttggag aggagctgag cttcgctcgg
     2881 gactgcagca gctccggcgc taagccgatc ctcgtggtcc tggatccaac agaaaatgac
     2941 aagctcactg gattgcaagc cgcataccgc gaggttggag gcgccgcata tgtgggggat
     3001 gccgcctggg ctcacttaga ggacgaggct ggtgctacca tggccagttt tatagagcgc
     3061 tatgtcaggg tgccagtagc atcagtcagt tccttcgaaa gggtcattga gggagatgcc
     3121 acaaagcgct ccttgatcct gcaggacctc caggcccgac tcgatggtaa tgagcttaca
     3181 atctccctgg gaggccatca gcggttggtg gaaaggcatg aggaccagag tctcgcagcg
     3241 gacggcgatg acgattcaga gctctacccc tatgatgtgc cagattacgc ctgagctctg
     3301 agcggccgct ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     3361 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3421 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3481 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3541 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3601 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3661 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3721 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3781 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3841 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3901 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3961 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4021 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4081 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4141 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4201 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4261 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4321 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4381 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4441 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4501 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4561 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4621 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4681 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4741 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4801 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4861 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4921 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4981 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     5041 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5101 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5161 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5221 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5281 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5341 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5401 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5461 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5521 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5581 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5641 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5701 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5761 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5821 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5881 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5941 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6001 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6061 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6121 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6181 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6241 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6301 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6361 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6421 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6481 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6541 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6601 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6661 gttaagggat tttggtcatg agattatcaa aaaggatctt cacct
//
