LOCUS       sequence-173701-        7052 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 7052)
  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 7052)
  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..7052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     738..757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      776..785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             785..826
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             827..1573
                     /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(2480..2498)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2481..2498)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2518..2535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2524..2748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2794..3222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2881..2900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3091..3112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3231..3251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3236..3565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3416..3551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3478..3497
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3632..4426
                     /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(3686..3705)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4296..4315
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4600..4721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4637..4656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4691..4710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4770..4786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4770..4786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4783..4805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4794..4810
                     /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(4818..4824,4825..4842,4843..4848))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4863..4884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5001..5018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5172..5760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5252..5271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5931..6722,6723..6791))
                     /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     6554..6573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6792..6896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6971..6990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg cggccgccac
      781 catgggcaag cccatcccca accccctgct gggcctggac agcaccggaa agtcttaccc
      841 aactgtgagt gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt
      901 catcgctgag aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac
      961 ctttgacaag ggcacgaaga ccggtggacc cttcggaacc atcaagcacc ctgccgaact
     1021 ggctcacagc gctaacaacg gtcttgacat cgctgttagg cttttggagc cactcaaggc
     1081 ggagttccct attttgagct acgccgattt ctaccagttg gctggcgttg ttgccgttga
     1141 ggtcacgggt ggacctaagg ttccattcca ccctggaaga gaggacaagc ctgagccacc
     1201 accagagggt cgcttgcccg atcccactaa gggttctgac catttgagag atgtgtttgg
     1261 caaagctatg gggcttactg accaagatat cgttgctcta tctgggggtc acactattgg
     1321 agctgcacac aaggagcgtt ctggatttga gggtccctgg acctctaatc ctcttatttt
     1381 cgacaactca tacttcacgg agttgttgag tggtgagaag gaaggtctcc ttcagctacc
     1441 ttctgacaag gctcttttgt ctgaccctgt attccgccct ctcgttgata aatatgcagc
     1501 ggacgaagat gccttctttg ctgattacgc tgaggctcac caaaagcttt ccgagcttgg
     1561 gtttgctgat gccaagctag caatggagcg tccgcaaccc gacagcatgc cccaggattt
     1621 gtcagaggcc ctgaaggagg ccaccaagga ggtgcacacc caggcagaga atgctgagtt
     1681 catgaggaac tttcagaagg gccaggtgac ccgagacggc ttcaagctgg tgatggcctc
     1741 cctgtaccac atctatgtgg ccctggagga ggagattgag cgcaacaagg agagcccagt
     1801 cttcgcccct gtctacttcc cagaagagct gcaccgcaag gctgccctgg agcaggacct
     1861 ggccttctgg tacgggcccc gctggcagga ggtcatcccc tacacaccag ccatgcagca
     1921 ctatgtgaag cggctccacg aggtggggcg cacagagccc gagctgctgg tggcccacgc
     1981 ctacacccgc tacctgggtg acctgtctgg gggccaggtg ctcaaaaaga ttgcccagaa
     2041 agccctggac ctgcccagct ctggcgaggg cctggccttc ttcaccttcc ccaacattgc
     2101 cagtgccacc aagttcaagc agctctaccg ctcccgcatg aactccctgg agatgactcc
     2161 cgcagtcagg cagagggtga tagaagaggc caagactgcg ttcctgctca acatccagct
     2221 ctttgaggag ttgcaggagc tgctgaccca tgacaccaag gaccagagcc cctcacgggc
     2281 accagggctt cgccagcggg ccagcaacaa agtgcaagat tctgcccccg tggagactcc
     2341 cagagggaag cccccactca acacccgctc ccaggctccg cttctccgat gggtccttac
     2401 actcagcttt ctggtggcga cagttgctgt agggctttat gccatgtaat gactcgagca
     2461 tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct
     2521 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2581 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2641 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2701 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     2761 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     2821 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2881 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2941 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3001 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3061 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3121 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3181 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3241 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3301 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3361 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3421 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3481 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3541 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3601 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3661 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3721 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3781 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3841 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3901 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3961 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4021 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4081 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4141 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4201 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4261 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4321 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4381 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4441 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4501 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4561 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4621 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4681 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4741 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4801 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4861 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4921 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4981 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5041 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5101 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5161 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5221 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5281 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5341 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5401 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5461 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5521 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5581 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5641 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5701 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5761 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5821 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5881 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5941 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6001 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6061 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6121 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6181 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6241 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6301 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6361 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6421 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6481 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6541 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6601 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6661 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6721 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6781 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6841 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6901 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     6961 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7021 gtgtgttgga ggtcgctgag tagtgcgcga gc
//
