LOCUS       sequence-324212-        5586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a nuclear exporting signal (NES) tagged with APEX2 and 
            3xflag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5586)
  AUTHORS   Bonet-Ponce L, Beilina A, Williamson CD, Lindberg E, Kluss JH, 
            Saez-Atienzar S, Landeck N, Kumaran R, Mamais A, Bleck CKE, Li Y, 
            Cookson MR
  TITLE     LRRK2 mediates tubulation and vesicle sorting from lysosomes.
  JOURNAL   Sci Adv. 2020 Nov 11;6(46). pii: 6/46/eabb2454. doi: 
            10.1126/sciadv.abb2454. Print 2020 Nov.
  PUBMED    33177079
REFERENCE   2  (bases 1 to 5586)
  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: "Sci Adv."; 
            date: "2020-11-11"; volume: "6(46). pii: 6/46/eabb2454. doi"; pages:
            " 10.1126/sciadv.abb2454. Print 2020 Nov"
COMMENT     Sequence Label: 3xflag-APEX2-NES
FEATURES             Location/Qualifiers
     source          1..5586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             931..996
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1033..1057
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1072..1818
                     /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             1825..1851
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     protein_bind    complement(1869..1893)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1927..2549
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2126..2145)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2573..2593)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2578..2907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2758..2893
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2945..2964)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2946..2964)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2978..2994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2978..2994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2991..3013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3002..3018
                     /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(3026..3032,3033..3050,3051..3056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3071..3092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3209..3226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3380..3968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3460..3479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4139..4930,4931..4999))
                     /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     4762..4781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5000..5104)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5131..5559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5218..5237)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5428..5449
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagcttg cggccgcgaa ttcatcgata
     1021 gatctgatat caacaagttt gtacaaaaaa gcaggctccg aattcgccct tggaaagtct
     1081 tacccaactg tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga
     1141 ggcttcatcg ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct
     1201 ggaacctttg acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc
     1261 gaactggctc acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc
     1321 aaggcggagt tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc
     1381 gttgaggtca cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag
     1441 ccaccaccag agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg
     1501 tttggcaaag ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact
     1561 attggagctg cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt
     1621 attttcgaca actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag
     1681 ctaccttctg acaaggctct tttgtctgac cctgtattcc gccctctcgt tgacaaatat
     1741 gcagcggacg aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag
     1801 cttgggtttg ctgatgccct gcagctgcct cccctggagc gcctgaccct ggactaaggg
     1861 cgaattcgac ccagctttct tgtacaaagt ggttgatatc ggtaccagtc gactctagag
     1921 gatcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     1981 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     2041 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     2101 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     2161 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     2221 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     2281 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     2341 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     2401 cttctgattt taaaataact ataccagcag gaggacgtcc agacacagca taggctacct
     2461 ggccatgccc aaccggtggg acatttgagt tgcttgcttg gcactgtcct ctcatgcgtt
     2521 gggtccactc agtagatgcc tgttgaattg ggtacgcggc cagcttggct gtggaatgtg
     2581 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2641 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2701 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2761 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2821 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     2881 ttttttggag gcctaggctt ttgcaaaaag ctcctcgagg aactgaaaaa ccagaaagtt
     2941 aattccctat agtgagtcgt attaaattcg taatcatgtc atagctgttt cctgtgtgaa
     3001 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3061 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3121 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3181 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3241 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3301 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3361 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3421 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3481 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3541 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3601 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3721 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3781 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3841 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3901 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3961 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4021 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4081 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4741 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5101 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
     5161 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5221 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5281 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5341 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5401 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5461 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5521 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5581 caattt
//
