LOCUS       40924_29471        8553 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Cytosolic APEX2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8553)
  AUTHORS   Hung V, Lam SS, Udeshi ND, Svinkina T, Guzman G, Mootha VK, Carr SA,
            Ting AY
  TITLE     Proteomic mapping of cytosol-facing outer mitochondrial and ER 
            membranes in living human cells by proximity biotinylation.
  JOURNAL   Elife. 2017 Apr 25;6. pii: e24463. doi: 10.7554/eLife.24463.
  PUBMED    28441135
REFERENCE   2  (bases 1 to 8553)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8553)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2017-04-25"; pages: "
            10.7554/eLife.24463"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(154..742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(916..1773)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1774..1878)
                     /label=AmpR promoter
     rep_origin      complement(1904..2359)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2501..2517
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2527..2545
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(2566..2701)
                     /direction=LEFT
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    complement(2728..2862)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     LTR             complement(2940..3173)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    complement(3245..3833)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     CDS             complement(3875..3916)
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     protein_bind    3918..3942
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(3950..3976)
                     /codon_start=1
                     /label=NES
                     /note="nuclear export signal from the HIV Rev protein
                     (Fischer et al., 1995)"
                     /translation="LPPLERLTL"
     CDS             complement(3983..4729)
                     /codon_start=1
                     /label=APEX2
                     /note="soybean ascorbate peroxidase, engineered to serve as
                     a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             complement(4730..4753)
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     protein_bind    complement(4774..4798)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(4821..5024)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(5025..5328)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    complement(5445..5562)
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     CDS             complement(5622..6017)
                     /codon_start=1
                     /label=BSD
                     /note="blasticidin S deaminase"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(6029..6529)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             complement(6704..6748)
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     misc_feature    complement(6933..7166)
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    complement(7659..7784)
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     LTR             7831..8011
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(8012..8238)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     promoter        complement(8266..8284)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8305..8321)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8329..8345)
                     /label=lac operator
                     /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(8353..8383)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8398..8419)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(8536..8553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
       61 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
      421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
      481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
      541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
      601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
      721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
      781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
      841 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
      901 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
      961 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1021 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1081 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1141 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     1201 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     1261 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     1321 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     1381 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     1441 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     1501 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     1561 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     1621 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     1681 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     1741 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     1801 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     1861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     1921 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     1981 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2041 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2101 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2161 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2221 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2281 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2341 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2401 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2461 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     2521 cgcgcgtaat acgactcact atagggcgaa ttgggtacgt ccctaggcct ccaaaaaagc
     2581 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     2641 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     2701 tgggcggagt taggggcggg atagctagag ccagacatga taagatacat tgatgagttt
     2761 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2821 attgctttat ttgtaaccat tataagctgc aataaacaag ttcctctcac tctctgatat
     2881 tcatttcttt gcaagttata aatactgaat aataagatga catgaactac tatacgtact
     2941 gctagagatt ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca
     3001 caacagacgg gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag
     3061 tgggttccct agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc
     3121 agtacaagca aaaagcagat cttgtcttcg ttgggagtga attagccctt ccagtccccc
     3181 cttttctttt aaaaagtggc taagatctac agctgccttg taagtcattg gtcttaaagt
     3241 cgacgcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag gcgaagacgc
     3301 ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg attgagggcc
     3361 gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca ggcgagcagc
     3421 caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc caacagccga
     3481 gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aatagggagg
     3541 gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca accagtgggg
     3601 gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg ccacaactcc
     3661 tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa
     3721 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca
     3781 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attgtcgact
     3841 taacgcgcca ccggttagcg ctagctcatt actacgtaga atcgagaccg aggagagggt
     3901 tagggatagg cttaccaacc actttgtaca agaaagctgg gtcttagtcc agggtcaggc
     3961 gctccagggg aggcagctgc agggcatcag caaacccaag ctcggaaagc ttttggtgag
     4021 cctcagcgta atcagcaaag aaggcatctt cgtccgctgc atatttgtca acgagagggc
     4081 ggaatacagg gtcagacaaa agagccttgt cagaaggtag ctgaaggaga ccttccttct
     4141 caccactcaa caactccgtg aagtatgagt tgtcgaaaat aagaggatta gaggtccagg
     4201 gaccctcaaa tccagaacgc tccttgtgtg cagctccaat agtgtgaccc ccagatagag
     4261 caacgatatc ttggtcagta agccccatag ctttgccaaa cacatctctc aaatggtcag
     4321 aacccttagt gggatcgggc aagcgaccct ctggtggtgg ctcaggcttg tcctctcttc
     4381 cagggtggaa tggaacctta ggtccacccg tgacctcaac ggcaacaacg ccagccaact
     4441 ggtagaaatc ggcgtagctc aaaataggga actccgcctt gagtggctcc aaaagcctaa
     4501 cagcgatgtc aagaccgttg ttagcgctgt gagccagttc ggcagggtgc ttgatggttc
     4561 cgaagggtcc accggtcttc gtgcccttgt caaaggttcc agcagagtgg aatgccaaac
     4621 ggagcattag aggagcgcat ctcttctcag cgatgaagcc tctgagcttc ttcttcgcct
     4681 tctcaacggc gtcctggtaa tcagcactca cagttgggta agactttccc ttatcgtcgt
     4741 catccttgta gtccatggtt ctatctcctt cgaagcctgc ttttttgtac aaacttgttg
     4801 atcccgacag ttagccagag agctctgctt atatagacct cccaccgtac acgcctaccg
     4861 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     4921 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     4981 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     5041 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     5101 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     5161 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     5221 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     5281 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     5341 tatgggctat gaactaatga ccccgtaatt gattactatt aataaccccg ggccccctcg
     5401 agccggcgcc aaagtggatc tctgctgtcc ctgtaataaa cccgaaaatt ttgaattttt
     5461 gtaatttgtt tttgtaattc tttagtttgt atgtctgttg ctattatgtc tactattctt
     5521 tcccctgcac tgtacccccc aatcccccct tttcttttaa aattgtggat gaatactgcc
     5581 atttgtctca agatctagaa ttcttactac tgcagctgca ggccctccca cacataacca
     5641 gagggcagca attcacgaat cccaactgcc gtcggctgtc catcactgtc cttcactatg
     5701 gctttgatcc caggatgcag atcgagaagc acctgtcggc accgtccgca ggggctcaag
     5761 atgcccctgt tctcatttcc gatcgcgacg atacaagtca ggttgccagc tgccgcagca
     5821 gcagcagtgc ccagcaccac gagttctgca caaggtcccc cagtaaaatg atatacattg
     5881 acaccagtga agatgcggcc gtcgctagag agagctgcgc tggcgacgct gtagtcttca
     5941 gagatgggga tgctgttgat tgtagccgtt gctctttcaa tgagggtgga ttcttcttga
     6001 gacaaaggct tggccatggt ggtaccccct ggggagagag gtcggtgatt cggtcaacga
     6061 gggagccgac tgccgacgtg cgctccggag gcttgcagaa tgcggaacac cgcgcgggca
     6121 ggaacagggc ccacactacc gccccacacc ccgcctcccg caccgcccct tcccggccgc
     6181 tgctctcggc gcgccctgct gagcagccgc tattggccac agcccatcgc ggtcggcgcg
     6241 ctgccattgc tccctggcgc tgtccgtctg cgagggtact agtgagacgt gcggcttccg
     6301 tttgtcacgt ccggcacgcc gcgaaccgca aggaaccttc ccgacttagg ggcggagcag
     6361 gaagcgtcgc cggggggccc acaagggtag cggcgaagat ccgggtgacg ctgcgaacgg
     6421 acgtgaagaa tgtgcgagac ccagggtcgg cgccgctgcg tttcccggaa ccacgcccag
     6481 agcagccgcg tccctgcgca aacccagggc tgccttggaa aaggcgcaag ggtcccctcg
     6541 gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta
     6601 tagaaagtac agcaaaaact attcttaaac ctaccaagcc tcctactatc attatgaata
     6661 attttatata ccacagccaa tttgttatgt taaaccaatt ccacaaactt gcccatttat
     6721 ctaattccaa taattcttgt tcattctttt cttgctggtt ttgcgattct tcaattaagg
     6781 agtgtattaa gcttgtgtaa ttgttaattt ctctgtccca ctccatccag gtcgtgtgat
     6841 tccaaatctg ttccagagat ttattactcc aactagcatt ccaaggcaca gcagtggtgc
     6901 aaatgagttt tccagagcaa ccccaaatcc ccaggagctg ttgatccttt aggtatcttt
     6961 ccacagccag gattcttgcc tggagctgct tgatgcccca gactgtgagt tgcaacagat
     7021 gctgttgcgc ctcaatagcc ctcagcaaat tgttctgctg ctgcactata ccagacaata
     7081 attgtctggc ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt cctgctgctc
     7141 ccaagaaccc aaggaacaaa gctcctattc ccactgctct tttttctctc tgcaccactc
     7201 ttctctttgc cttggtgggt gctactccta atggttcaat ttttactact ttatatttat
     7261 ataattcact tctccaattg tccctcatat ctcctcctcc aggtctgaag atcagcggcc
     7321 gcttgctgtg cggtggtctt acttttgttt tgctcttcct ctatcttgtc taaagcttcc
     7381 ttggtgtctt ttatctctat cctttgatgc acacaataga gggttgctac tgtattatat
     7441 aatgatctaa gttcttctga tcctgtctga agggatggtt gtagctgtcc cagtatttgt
     7501 ctacagcctt ctgatgtttc taacaggcca ggattaactg cgaatcgttc tagctccctg
     7561 cttgcccata ctatatgttt taatttatat tttttctttc cccctggcct taaccgaatt
     7621 ttttcccatc gcgatctaat tctcccccgc ttaatactga cgctctcgca cccatctctc
     7681 tccttctagc ctccgctagt caaaattttt ggcgtactca ccagtcgccg cccctcgcct
     7741 cttgccgtgc gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc tggtttccct
     7801 ttcgctttca agtccctgtt cgggcgccac tgctagagat tttccacact gactaaaagg
     7861 gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca
     7921 ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc
     7981 aggctcagat ctggtctaac cagagagacc cgtttatgta tcgagctagg cacttaaata
     8041 caatatctct gcaatgcggc aattcagtgg ttcgtccaat ccatgtcaga cccgtctgtt
     8101 gccttcctaa taaggcacga tcgtaccacc ttacttccac caatcggcat gcacggtgct
     8161 ttttctctcc ttgtaaggca tgttgctaac tcatcgttac catgttgcaa gactacaaga
     8221 gtattgcata agactacatt aagcttgcag ctccagcttt tgttcccttt agtgagggtt
     8281 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     8341 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     8401 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     8461 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     8521 gcgctcttcc gcttcctcgc tcactgactc gct
//
