LOCUS       sequence-485229-        5660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human APOX in mitochondrial matrix.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5660)
  AUTHORS   Fang S, Acevedo LD, Solivais AJ, Zhou X, Delfosse ES, Frey BL, Smith
            LM, Martell JD
  TITLE     Directed Evolution of APOX for Proximity Labeling Using Phenols with
            High Redox Potentials
  JOURNAL   ChemRxiv. 2025; doi:10.26434/chemrxiv-2025-07zmn
REFERENCE   2  (bases 1 to 5660)
  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: "ChemRxiv.";
            date: "2025"; volume: " doi"; pages: "10.26434/chemrxiv-2025-07zm"
COMMENT     Sequence Label: CAG-MITO-APOX
FEATURES             Location/Qualifiers
     source          1..5660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..663
                     /label=chicken beta-actin promoter
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2567..2590
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2682..2701)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2747..2802
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2801..2820)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3163..3179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3163..3179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3176..3198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3187..3203
                     /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(3211..3217,3218..3235,3236..3241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3256..3277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3336..3531
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3382..3517
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3537..3671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3574..3593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3628..3647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3739..3756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3910..4498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3990..4009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4669..5460,5461..5529))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5292..5311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5530..5634)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgagggggg gggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
      541 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgggggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attccgccac
     1741 catgctggcc acccgcgtgt tcagcctggt gggcaagcgc gccatcagca ccagcgtgtg
     1801 cgtgcgcgcc cacgctagcg ggaaatcata cccaacagtg tccgcagact accaggatgc
     1861 cgtggagaaa gccaagaaga agctgagagg gtttatcgca gaaaagaggt gcgcacctct
     1921 gatgctgaga ctggctgtcc acagcgcagg cacctttgac aagggaacca aaacaggcgg
     1981 accctttgga acaatcaagc accctgctga actggcacat attgccaaca atgggctgga
     2041 catcgccgtg cggctgctgg aacctctgaa agcagagttc ccaattctgt cctacgccga
     2101 tttttatcag ctggcaggag tggtcgctgt ggaggtcact gggggcccca aggtgccttt
     2161 ccacccagga cgggaggaca aaccagaacc acctccagag gggcgcctgc cagatccgac
     2221 aaagggctcc gaccatctgc gagatgtgtt tgggaaagct atgggcctga ctgaccagga
     2281 tatcgtcgca ctgtctggag ggcacaccat tggcgccgct cataaggaaa ggtcaggctt
     2341 cgagggaccc tggacaagca accccctgat tttcgacaat tcttacttta ctgaactgct
     2401 gagtggagag aaggaagggc tgctgcagct gcccagcgac aaggccctgc tgtccgatcc
     2461 cgtgttcaga cctctggtcg ataagtatgc agccgacgag gatgcttttt tcgcagattt
     2521 cgcagaagca tatcagaagc tgtcagaact gggatttgcc gacgccgact acaaggatga
     2581 cgacgataag taatgagcgg ccgcactcct caggtgcagg ctgcctatca gaaggtggtg
     2641 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
     2701 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     2761 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     2821 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     2881 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     2941 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     3001 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     3061 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     3121 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
     3181 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3241 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3301 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     3361 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3421 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3481 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     3541 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3601 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3661 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3721 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3781 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3841 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3901 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3961 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4021 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4081 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4141 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4201 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4261 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4321 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4381 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4441 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4501 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4561 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4621 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4681 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4741 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4801 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4861 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4921 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4981 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5041 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5101 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5161 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5221 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5281 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5341 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5401 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5461 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5521 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5581 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5641 tccccgaaaa gtgccacctg
//
