LOCUS       sequence-419150-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mitochondrial localized ApaLI with D211A, 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-ApaLI*-HA
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             967..1041
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2200..2226
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2260..2278)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2261..2278)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2298..2315)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2304..2528
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2574..3002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2661..2680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2871..2892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3011..3031)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3016..3345
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3196..3331
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3258..3277
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3412..4206
                     /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(3466..3485)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4076..4095
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4380..4501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4417..4436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4471..4490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4550..4566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4550..4566)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4563..4585)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4574..4590
                     /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(4598..4604,4605..4622,4623..4628))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4643..4664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4781..4798)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4952..5540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5032..5051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5711..6502,6503..6571))
                     /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     6334..6353
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6572..6676)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctagctt
      901 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattcg ggtactccgt
      961 gccatcatgt ccgtcctgac gccgctgctg ctgcggggct tgacaggctc ggcccggcgg
     1021 ctcccagtgc cgcgcgccaa gatccattcg ttgccgccgg aggggaagct tatgaccaca
     1081 cgacagcgct tatctgctga gagatctcag cagcttaccc gtctgctcac tatcactaaa
     1141 accgctaaca tgcgggcctt aatggaggct agcgaactcg ctaaagtaat tgccctggtg
     1201 gcggttgaca taggtaagtc agacgagatg gccagagctt tccccgtgct gtggccaaaa
     1261 atcagtcctc agcaagaata ttacgcaaca gcggttgact ggttcaccaa ccccgatgaa
     1321 accgtcacct ctttcgatgt ggtggatatg ctagatgccg gcacttccct ggaccaagac
     1381 ttcatgactt atctgaaatg tctaactgaa ctgcacaagc gacggagaaa atacggcctg
     1441 atcctgcaga ggcagcctct accaacgatg gtgcaggttt cgccccgagc actgatggaa
     1501 tacggacctg actttccgcc cgaggcactg gctagctggc ttacctggcg gaagtttttt
     1561 tacgaccttg acaacaggtc tgcccaggaa acagggtact tgttcgaacc cattctggcc
     1621 gccgccatcg gaggcgaggc aaaatctgcc agagagcgtg tcgtaagacg caccgacgat
     1681 ccaacgaagg gcaggcaggt ggcctgctgg aaggtgctcc ctgatggtac gcctctggct
     1741 tatgcgttgg cgcttcgggt gacaattgca gcgagcgggc aaggcagatt tggagaggag
     1801 ctgagcttcg ctcgggactg cagcagctcc ggcgctaagc cgatcctcgt ggtcctggat
     1861 ccaacagaaa atgacaagct cactggattg caagccgcat accgcgaggt tggaggcgcc
     1921 gcatatgtgg gggatgccgc ctgggctcac ttagaggacg aggctggtgc taccatggcc
     1981 agttttatag agcgctatgt cagggtgcca gtagcatcag tcagttcctt cgaaagggtc
     2041 attgagggag atgccacaaa gcgctccttg atcctgcagg acctccaggc ccgactcgat
     2101 ggtaatgagc ttacaatctc cctgggaggc catcagcggt tggtggaaag gcatgaggac
     2161 cagagtctcg cagcggacgg cgatgacgat tcagagctct acccctatga tgtgccagat
     2221 tacgcctgag ctctgagcgg ccgctctaga gggccctatt ctatagtgtc acctaaatgc
     2281 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2341 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2401 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2461 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2521 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2581 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2641 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2701 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2761 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2821 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2881 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2941 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3001 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3061 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3121 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3181 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3241 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3301 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3361 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3421 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3481 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3541 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3601 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3661 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3721 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3781 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3841 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3901 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3961 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4021 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4081 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4141 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4201 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4261 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4321 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4381 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4441 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4501 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4561 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4621 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4681 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4741 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4801 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4861 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4921 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4981 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5281 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5341 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5401 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5461 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5521 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5581 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5641 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5701 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5761 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5821 catctggccc cagtgctgca atgataccgc gagacccacc cttggaggct ccagatttat
     5881 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5941 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6001 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6061 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6121 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6181 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6241 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6301 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6361 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6421 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6481 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6541 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6601 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6661 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacg
//
