LOCUS       sequence-173675-        6476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed N-term tagged APEX2 on Sec61B in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6476)
  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 6476)
  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-Sec61B
FEATURES             Location/Qualifiers
     source          1..6476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        116..495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        496..699
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     650..670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     744..763
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        744..762
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      782..791
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             791..832
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             833..1579
                     /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             1589..1879
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     promoter        complement(1910..1928)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1911..1928)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1948..1965)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1954..2178
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2224..2652
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2311..2330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2521..2542
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2661..2681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2666..2995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2846..2981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2908..2927
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3062..3856
                     /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(3116..3135)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3726..3745
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4030..4151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4067..4086)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4121..4140
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4200..4216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4200..4216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4213..4235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4224..4240
                     /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(4248..4254,4255..4272,4273..4278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4293..4314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4431..4448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4602..5190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4682..4701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5361..6152,6153..6221))
                     /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     5984..6003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6222..6326)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6401..6420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
       61 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
      121 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      181 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      241 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      301 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      361 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      421 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      481 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      541 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      601 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      661 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
      721 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agcttgcggc
      781 cgccaccatg ggcaagccca tccccaaccc cctgctgggc ctggacagca ccggaaagtc
      841 ttacccaact gtgagtgctg attaccagga cgccgttgag aaggcgaaga agaagctcag
      901 aggcttcatc gctgagaaga gatgcgctcc tctaatgctc cgtttggcat tccactctgc
      961 tggaaccttt gacaagggca cgaagaccgg tggacccttc ggaaccatca agcaccctgc
     1021 cgaactggct cacagcgcta acaacggtct tgacatcgct gttaggcttt tggagccact
     1081 caaggcggag ttccctattt tgagctacgc cgatttctac cagttggctg gcgttgttgc
     1141 cgttgaggtc acgggtggac ctaaggttcc attccaccct ggaagagagg acaagcctga
     1201 gccaccacca gagggtcgct tgcccgatcc cactaagggt tctgaccatt tgagagatgt
     1261 gtttggcaaa gctatggggc ttactgacca agatatcgtt gctctatctg ggggtcacac
     1321 tattggagct gcacacaagg agcgttctgg atttgagggt ccctggacct ctaatcctct
     1381 tattttcgac aactcatact tcacggagtt gttgagtggt gagaaggaag gtctccttca
     1441 gctaccttct gacaaggctc ttttgtctga ccctgtattc cgccctctcg ttgataaata
     1501 tgcagcggac gaagatgcct tctttgctga ttacgctgag gctcaccaaa agctttccga
     1561 gcttgggttt gctgatgcca agctagcaat gcctggtccg acccccagtg gcactaacgt
     1621 gggatcctca gggcgctctc ccagcaaagc agtggccgcc cgggcggcgg gatccactgt
     1681 ccggcagagg aaaaatgcca gctgtgggac aaggagtgca ggccgcacaa cctcggcagg
     1741 caccgggggg atgtggcgat tctacacaga agattcacct gggctcaaag ttggccctgt
     1801 tccagtattg gttatgagtc ttctgttcat cgcttctgta tttatgttgc acatttgggg
     1861 caagtacact cgttcgtaat gactcgagca tgcatctaga gggccctatt ctatagtgtc
     1921 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1981 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2041 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2101 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2161 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2221 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2281 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2341 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2401 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2461 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2521 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2581 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2641 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2701 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2761 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2821 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2881 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     2941 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3001 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3061 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3121 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3181 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3241 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3301 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3361 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3421 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3481 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3541 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3601 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3661 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3721 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3781 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3841 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3901 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3961 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4021 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4081 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4141 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4201 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4261 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4381 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4441 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4501 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4561 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4621 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4681 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4741 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4801 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4861 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4921 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4981 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5041 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5101 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5161 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5221 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5281 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5341 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5401 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5461 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5521 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5581 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5641 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5701 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5761 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5821 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5881 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5941 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6001 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6061 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6121 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6181 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6241 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6301 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6361 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     6421 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgc
//
