LOCUS       sequence-173681-        7049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed C-term tagged APEX2 on HMOX1 in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7049)
  AUTHORS   Lee SY, Kang MG, Park JS, Lee G, Ting AY, Rhee HW
  TITLE     APEX Fingerprinting Reveals the Subcellular Localization of Proteins
            of Interest.
  JOURNAL   Cell Rep. 2016 May 11. pii: S2211-1247(16)30508-3. doi: 
            10.1016/j.celrep.2016.04.064.
  PUBMED    27184847
REFERENCE   2  (bases 1 to 7049)
  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: "Cell Rep. 
            2016 May 11. pii: S2211-1247(16)30508-3. doi: 
            10.1016/j.celrep.2016.04.064."
COMMENT     Sequence Label: pCDNA3_HMOX1-V5-APEX2
FEATURES             Location/Qualifiers
     source          1..7049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     717..736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        717..735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      755..764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1634..1675
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1676..2422
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     promoter        complement(2456..2474)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2457..2474)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2494..2511)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2500..2724
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2770..3198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2857..2876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3067..3088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3207..3227)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3212..3541
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3392..3527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3454..3473
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3608..4402
                     /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(3662..3681)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4272..4291
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4576..4697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4613..4632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4667..4686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4746..4762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4746..4762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4759..4781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4770..4786
                     /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(4794..4800,4801..4818,4819..4824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4839..4860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4977..4994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5148..5736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5228..5247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5907..6698,6699..6767))
                     /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     6530..6549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6768..6872)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6947..6966)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
       61 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      721 acgactcact atagggagac ccaagcttgc ggccgccacc atggagcgtc cgcaacccga
      781 cagcatgccc caggatttgt cagaggccct gaaggaggcc accaaggagg tgcacaccca
      841 ggcagagaat gctgagttca tgaggaactt tcagaagggc caggtgaccc gagacggctt
      901 caagctggtg atggcctccc tgtaccacat ctatgtggcc ctggaggagg agattgagcg
      961 caacaaggag agcccagtct tcgcccctgt ctacttccca gaagagctgc accgcaaggc
     1021 tgccctggag caggacctgg ccttctggta cgggccccgc tggcaggagg tcatccccta
     1081 cacaccagcc atgcagcact atgtgaagcg gctccacgag gtggggcgca cagagcccga
     1141 gctgctggtg gcccacgcct acacccgcta cctgggtgac ctgtctgggg gccaggtgct
     1201 caaaaagatt gcccagaaag ccctggacct gcccagctct ggcgagggcc tggccttctt
     1261 caccttcccc aacattgcca gtgccaccaa gttcaagcag ctctaccgct cccgcatgaa
     1321 ctccctggag atgactcccg cagtcaggca gagggtgata gaagaggcca agactgcgtt
     1381 cctgctcaac atccagctct ttgaggagtt gcaggagctg ctgacccatg acaccaagga
     1441 ccagagcccc tcacgggcac cagggcttcg ccagcgggcc agcaacaaag tgcaagattc
     1501 tgcccccgtg gagactccca gagggaagcc cccactcaac acccgctccc aggctccgct
     1561 tctccgatgg gtccttacac tcagctttct ggtggcgaca gttgctgtag ggctttatgc
     1621 catgaagcta gcaggcaagc ccatccccaa ccccctgctg ggcctggaca gcaccggaaa
     1681 gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga agaagaagct
     1741 cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg cattccactc
     1801 tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca tcaagcaccc
     1861 tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc ttttggagcc
     1921 actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg ctggcgttgt
     1981 tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag aggacaagcc
     2041 tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc atttgagaga
     2101 tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat ctgggggtca
     2161 cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga cctctaatcc
     2221 tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg aaggtctcct
     2281 tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc tcgttgataa
     2341 atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc aaaagctttc
     2401 cgagcttggg tttgctgatg cctaatgact cgagcatgca tctagagggc cctattctat
     2461 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     2521 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2581 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2641 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2701 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2761 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2821 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2881 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2941 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3001 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3061 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3121 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3181 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3241 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3301 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3361 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3421 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3481 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3541 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     3601 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3661 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3721 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3781 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3841 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3901 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3961 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4021 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4081 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4141 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4201 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4261 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4321 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4381 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4441 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4501 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4561 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4621 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4681 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4741 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4801 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4861 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4921 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4981 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5041 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5101 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5161 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5221 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5281 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5341 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5401 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5461 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5521 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5581 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5641 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5701 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5761 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5821 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5881 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5941 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6001 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6061 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6121 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6181 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6241 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6301 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6361 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6421 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6481 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6541 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6601 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6661 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6721 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6781 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6841 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6901 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6961 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     7021 gcgcgagcaa aatttaagct acaacaagg
//
