LOCUS       sequence-173674-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed C-term tagged APEX2 on Sec61B in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  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 6473)
  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_Sec61B-V5-APEX2
FEATURES             Location/Qualifiers
     source          1..6473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        106..485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        486..689
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     640..660
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     734..753
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        734..752
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             778..1065
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     CDS             1075..1116
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1117..1863
                     /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(1897..1915)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1898..1915)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1935..1952)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1941..2165
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2211..2639
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2298..2317)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2508..2529
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2648..2668)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2653..2982
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2833..2968
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2895..2914
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3049..3843
                     /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(3103..3122)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3713..3732
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4017..4138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4054..4073)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4108..4127
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4187..4203)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4187..4203)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4200..4222)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4211..4227
                     /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(4235..4241,4242..4259,4260..4265))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4280..4301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4418..4435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4589..5177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4669..4688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5348..6139,6140..6208))
                     /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     5971..5990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6209..6313)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6388..6407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
       61 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
      121 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      181 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      241 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      301 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      361 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      421 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      481 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      541 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      601 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      661 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      721 ggcttatcga aattaatacg actcactata gggagaccca agcttgcggc cgccaccatg
      781 cctggtccga cccccagtgg cactaacgtg ggatcctcag ggcgctctcc cagcaaagca
      841 gtggccgccc gggcggcggg atccactgtc cggcagagga aaaatgccag ctgtgggaca
      901 aggagtgcag gccgcacaac ctcggcaggc accgggggga tgtggcgatt ctacacagaa
      961 gattcacctg ggctcaaagt tggccctgtt ccagtattgg ttatgagtct tctgttcatc
     1021 gcttctgtat ttatgttgca catttggggc aagtacactc gttcgaagct agcaggcaag
     1081 cccatcccca accccctgct gggcctggac agcaccggaa agtcttaccc aactgtgagt
     1141 gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt catcgctgag
     1201 aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac ctttgacaag
     1261 ggcacgaaga ccggtggacc cttcggaacc atcaagcacc ctgccgaact ggctcacagc
     1321 gctaacaacg gtcttgacat cgctgttagg cttttggagc cactcaaggc ggagttccct
     1381 attttgagct acgccgattt ctaccagttg gctggcgttg ttgccgttga ggtcacgggt
     1441 ggacctaagg ttccattcca ccctggaaga gaggacaagc ctgagccacc accagagggt
     1501 cgcttgcccg atcccactaa gggttctgac catttgagag atgtgtttgg caaagctatg
     1561 gggcttactg accaagatat cgttgctcta tctgggggtc acactattgg agctgcacac
     1621 aaggagcgtt ctggatttga gggtccctgg acctctaatc ctcttatttt cgacaactca
     1681 tacttcacgg agttgttgag tggtgagaag gaaggtctcc ttcagctacc ttctgacaag
     1741 gctcttttgt ctgaccctgt attccgccct ctcgttgata aatatgcagc ggacgaagat
     1801 gccttctttg ctgattacgc tgaggctcac caaaagcttt ccgagcttgg gtttgctgat
     1861 gcctaatgac tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag
     1921 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1981 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2041 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2101 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2161 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2221 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2281 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2341 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2401 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2461 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2521 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2581 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     2641 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2701 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2761 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2821 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2881 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     2941 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     3001 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3061 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3121 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3181 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     3241 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3301 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3361 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3421 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3481 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3541 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     3601 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3661 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3721 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3781 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3841 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3901 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3961 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4021 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4081 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4141 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4201 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4261 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4321 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4381 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4441 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4501 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4561 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4621 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4681 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4741 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4801 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4861 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4921 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4981 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5041 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5101 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5161 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5221 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5281 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5341 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5401 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5461 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5521 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5581 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5641 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5701 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5761 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5821 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5881 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5941 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6001 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6061 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6121 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6181 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6241 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6301 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     6361 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6421 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaa
//
