LOCUS       sequence-166869-        7053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed N-term tagged APEX2 on HMOX1 in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7053)
  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 7053)
  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_V5-APEX2-HMOX1
FEATURES             Location/Qualifiers
     source          1..7053
                     /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..951
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             952..1698
                     /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(2605..2623)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2606..2623)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2643..2660)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2649..2873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2919..3347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3006..3025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3216..3237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3356..3376)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3361..3691
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3542..3677
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3604..3623
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3758..4552
                     /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(3812..3831)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4422..4441
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4726..4847
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4763..4782)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4817..4836
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4896..4912)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4896..4912)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4909..4931)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4920..4936
                     /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(4944..4950,4951..4968,4969..4974))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4989..5010
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5127..5144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6057..6848,6849..6917))
                     /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     6680..6699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6918..7022)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcaagcccat ccccaacccc ctgctgggcc tggacagcac cggaaagtct
      961 tacccaactg tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga
     1021 ggcttcatcg ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct
     1081 ggaacctttg acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc
     1141 gaactggctc acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc
     1201 aaggcggagt tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc
     1261 gttgaggtca cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag
     1321 ccaccaccag agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg
     1381 tttggcaaag ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact
     1441 attggagctg cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt
     1501 attttcgaca actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag
     1561 ctaccttctg acaaggctct tttgtctgac cctgtattcc gccctctcgt tgataaatat
     1621 gcagcggacg aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag
     1681 cttgggtttg ctgatgccaa gctagcaatg gagcgtccgc aacccgacag catgccccag
     1741 gatttgtcag aggccctgaa ggaggccacc aaggaggtgc acacccaggc agagaatgct
     1801 gagttcatga ggaactttca gaagggccag gtgacccgag acggcttcaa gctggtgatg
     1861 gcctccctgt accacatcta tgtggccctg gaggaggaga ttgagcgcaa caaggagagc
     1921 ccagtcttcg cccctgtcta cttcccagaa gagctgcacc gcaaggctgc cctggagcag
     1981 gacctggcct tctggtacgg gccccgctgg caggaggtca tcccctacac accagccatg
     2041 cagcactatg tgaagcggct ccacgaggtg gggcgcacag agcccgagct gctggtggcc
     2101 cacgcctaca cccgctacct gggtgacctg tctgggggcc aggtgctcaa aaagattgcc
     2161 cagaaagccc tggacctgcc cagctctggc gagggcctgg ccttcttcac cttccccaac
     2221 attgccagtg ccaccaagtt caagcagctc taccgctccc gcatgaactc cctggagatg
     2281 actcccgcag tcaggcagag ggtgatagaa gaggccaaga ctgcgttcct gctcaacatc
     2341 cagctctttg aggagttgca ggagctgctg acccatgaca ccaaggacca gagcccctca
     2401 cgggcaccag ggcttcgcca gcgggccagc aacaaagtgc aagattctgc ccccgtggag
     2461 actcccagag ggaagccccc actcaacacc cgctcccagg ctccgcttct ccgatgggtc
     2521 cttacactca gctttctggt ggcgacagtt gctgtagggc tttatgccat gtaatgactc
     2581 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2641 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2701 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2761 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2821 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2881 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3061 agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc
     3301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3361 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag
     3421 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3481 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3541 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3601 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3661 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3721 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3781 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3841 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3901 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3961 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4021 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4081 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4141 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4201 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4261 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4321 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4381 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4441 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4501 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4561 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4621 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4681 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4741 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4801 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4861 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4921 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4981 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5041 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5101 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6181 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6361 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6661 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7021 cgcacatttc cccgaaaagt gccacctgac gtc
//
