LOCUS       sequence-485070-        6158 bp    DNA     circular SYN 29-AUG-2025
DEFINITION  Expresses human APOX in cytosol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6158)
  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 6158)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 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: pCDNA3-APOX
FEATURES             Location/Qualifiers
     source          1..6158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..933
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1711..1729)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1712..1729)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1749..1766)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1755..1979
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2025..2453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2322..2343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2462..2482)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2467..2796
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2647..2782
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2709..2728
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2863..3657
                     /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(2917..2936)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3527..3546
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3831..3952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3868..3887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3922..3941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4001..4017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4001..4017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4014..4036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4025..4041
                     /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(4049..4055,4056..4073,4074..4079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4094..4115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4232..4249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4403..4991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4483..4502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5162..5953,5954..6022))
                     /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     5785..5804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6023..6127)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg actacaagga tgacgacgat aaggggaaat catacccaac agtgtccgca
      961 gactaccagg atgccgtgga gaaagccaag aagaagctga gagggtttat cgcagaaaag
     1021 aggtgcgcac ctctgatgct gagactggct gtccacagcg caggcacctt tgacaaggga
     1081 accaaaacag gcggaccctt tggaacaatc aagcaccctg ctgaactggc acatattgcc
     1141 aacaatgggc tggacatcgc cgtgcggctg ctggaacctc tgaaagcaga gttcccaatt
     1201 ctgtcctacg ccgattttta tcagctggca ggagtggtcg ctgtggaggt cactgggggc
     1261 cccaaggtgc ctttccaccc aggacgggag gacaaaccag aaccacctcc agaggggcgc
     1321 ctgccagatc cgacaaaggg ctccgaccat ctgcgagatg tgtttgggaa agctatgggc
     1381 ctgactgacc aggatatcgt cgcactgtct ggagggcaca ccattggcgc cgctcataag
     1441 gaaaggtcag gcttcgaggg accctggaca agcaaccccc tgattttcga caattcttac
     1501 tttactgaac tgctgagtgg agagaaggaa gggctgctgc agctgcccag cgacaaggcc
     1561 ctgctgtccg atcccgtgtt cagacctctg gtcgataagt atgcagccga cgaggatgct
     1621 tttttcgcag atttcgcaga agcatatcag aagctgtcag aactgggatt tgccgacgcc
     1681 taactcgagc atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg
     1741 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1801 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1861 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1921 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     1981 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2041 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2101 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2161 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2221 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2281 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2341 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2401 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2461 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2521 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2581 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2641 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2701 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2761 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     2821 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2881 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2941 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3001 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3061 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3121 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3181 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3241 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3301 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3361 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     3421 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3481 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3541 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3601 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3661 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     3721 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3781 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3841 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3901 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3961 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4021 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4081 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4141 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4201 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4261 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4321 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4381 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4441 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4501 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4561 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4621 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4681 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4741 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4801 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4861 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4921 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4981 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5041 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5101 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5161 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5221 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5281 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5341 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5401 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5461 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5521 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5581 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5641 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5701 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5761 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5821 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5881 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5941 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6001 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6061 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6121 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
