LOCUS       sequence-419569-        6257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses APEX2 in mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6257)
  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 6257)
  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: mito-V5-APEX2
FEATURES             Location/Qualifiers
     source          1..6257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(630..648)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(631..648)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(668..685)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    674..898
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      944..1372
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1031..1050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1241..1262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1381..1401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1386..1715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1566..1701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1628..1647
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1782..2576
                     /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(1836..1855)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2446..2465
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2750..2871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2787..2806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2841..2860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2920..2936)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2920..2936)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2933..2955)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2944..2960
                     /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(2968..2974,2975..2992,2993..2998))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3013..3034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3151..3168)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3322..3910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3402..3421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4081..4872,4873..4941))
                     /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     4704..4723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4942..5046)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5121..5140)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5312..5691
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5692..5895
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5846..5866
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5940..5959
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5940..5958
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6065..6106
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(6107..6257,1..596)
                     /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"
ORIGIN
        1 agaccggtgg acccttcgga accatcaagc accctgccga actggctcac agcgctaaca
       61 acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc cctattttga
      121 gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta
      181 aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag ggtcgcttgc
      241 ctgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct atggggctta
      301 ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca cacaaggagc
      361 gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac tcatacttca
      421 cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac aaggctcttt
      481 tgtctgaccc tgtattccgc cctctcgttg ataaatatgc agcggacgaa gatgccttct
      541 ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct gatgcctaat
      601 gactcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc
      661 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
      721 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
      781 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
      841 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
      901 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
      961 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1021 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1081 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1141 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1201 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1261 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1321 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     1381 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     1441 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     1501 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     1561 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     1621 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     1681 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     1741 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     1801 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     1861 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     1921 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     1981 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     2041 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     2101 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     2161 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     2221 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     2281 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     2341 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     2401 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     2461 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     2521 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     2581 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     2641 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     2701 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     2761 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2821 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2881 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2941 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3001 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3061 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3121 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3181 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3241 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3301 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3361 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3421 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3481 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3541 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3601 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3661 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3721 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3781 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3841 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3901 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3961 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4021 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4081 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4141 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4201 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4261 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4321 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4381 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4441 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4501 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4561 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4621 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4681 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     4741 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4801 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4861 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4921 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4981 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5041 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     5101 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     5161 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     5221 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     5281 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     5341 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     5401 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     5461 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     5521 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     5581 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     5641 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     5701 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     5761 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     5821 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     5881 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
     5941 aatacgactc actataggga gacccaagct tgcggccgcc accatgctgg ccacccgcgt
     6001 gttcagcctg gtgggcaagc gcgccatcag caccagcgtg tgcgtgcgcg cccacaagga
     6061 tccaggcaag cccatcccca accccctgct gggcctggac agcaccggaa agtcttaccc
     6121 aactgtgagt gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt
     6181 catcgctgag aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac
     6241 ctttgacaag ggcacga
//
