LOCUS       sequence-327822-        6197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses APEX2 in the cytosol of mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6197)
  AUTHORS   Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, 
            Ting AY
  TITLE     Directed evolution of APEX2 for electron microscopy and proximity 
            labeling.
  JOURNAL   Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
  PUBMED    25419960
REFERENCE   2  (bases 1 to 6197)
  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: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     Sequence Label: pcDNA3 APEX2-NES
FEATURES             Location/Qualifiers
     source          1..6197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     743..762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        743..761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      781..790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             790..813
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             814..1560
                     /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"
     CDS             1567..1593
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1630..1648)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1631..1648)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1668..1685)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1674..1898
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1944..2372
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2031..2050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2241..2262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2381..2401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2386..2715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2566..2701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2628..2647
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2782..3576
                     /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(2836..2855)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3446..3465
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3750..3871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3787..3806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3841..3860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3920..3936)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3920..3936)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3933..3955)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3944..3960
                     /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(3968..3974,3975..3992,3993..3998))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4013..4034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4151..4168)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4322..4910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4402..4421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5081..5872,5873..5941))
                     /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     5704..5723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5942..6046)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6121..6140)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      721 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      781 gccaccatgg actacaagga tgacgacgat aagggaaagt cttacccaac tgtgagtgct
      841 gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag
      901 agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc
      961 acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct
     1021 aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt
     1081 ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga
     1141 cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc
     1201 ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg
     1261 cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag
     1321 gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac
     1381 ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct
     1441 cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga cgaagatgcc
     1501 ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc
     1561 ctgcagctgc ctcccctgga gcgcctgacc ctggactaat agctcgagca tgcatctaga
     1621 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     1681 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1741 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1801 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1861 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     1921 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1981 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2041 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2101 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2161 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2221 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2281 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2341 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2401 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2461 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2521 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2581 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2641 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2701 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2761 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2821 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2881 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2941 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3001 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3061 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3121 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3181 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3241 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3301 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3361 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3421 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3481 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3541 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3601 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3661 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3721 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3781 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3841 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3901 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3961 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4021 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4081 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4141 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4201 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4261 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4321 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4381 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4441 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4501 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4561 ccaagctggg ctgtgtgcac aaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4621 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4681 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4741 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4801 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4861 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4921 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4981 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5041 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5101 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5161 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5221 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5281 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5341 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5401 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5461 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5521 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5581 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5641 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5701 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5761 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5821 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5881 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5941 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6001 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6061 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     6121 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     6181 ggtcgctgag tagtgcg
//
