LOCUS       sequence-210087-        4108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for 5' tagging with APEX2 (includes 
            cytoplasmic GFP reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4108)
  AUTHORS   Ariotti N, Rae J, Giles N, Martel N, Sierecki E, Gambin Y, Hall TE, 
            Parton RG
  TITLE     Ultrastructural localisation of protein interactions using 
            conditionally stable nanobodies.
  JOURNAL   PLoS Biol. 2018 Apr 5;16(4):e2005473. doi: 
            10.1371/journal.pbio.2005473. eCollection 2018 Apr.
  PUBMED    29621251
REFERENCE   2  (bases 1 to 4108)
  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: "PLoS 
            Biol."; date: "2018-04-5"; volume: "16(4):e2005473. doi"; pages: " 
            10.1371/journal.pbio.2005473. eCollection 2018 Apr"
COMMENT     Sequence Label: pME-EGFP-P2A-APEX2-NS
FEATURES             Location/Qualifiers
     source          1..4108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(273..300)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(392..478)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     527..549
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     541..558
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     542..558
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    574..673
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      674..683
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(680..682,683..685,686..1396)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(680..682,683..685,686..1396)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(725..746)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(986..1005)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1333..1354
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1412..1468
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1487..2233
                     /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"
     protein_bind    complement(2238..2337)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2354..2373)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2355..2373)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2378..2394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2378..2394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2496..2515)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2507..3316
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2574..2593)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3463..4051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3952..3971
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
       61 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      121 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      181 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt
      241 accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct
      301 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
      361 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
      421 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
      481 gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt
      541 tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata
      601 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac
      661 aaaaaagcag gctgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      721 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      781 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      841 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
      901 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      961 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1021 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1081 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1141 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1201 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1261 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1321 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1381 ggacgagctg tacaagccta ggggcagcgg cgccaccaac ttcagcctgc tgaagcaggc
     1441 cggcgacgtg gaggagaacc ccggccccgc ccccggcagc ctcgagggaa agtcttaccc
     1501 aactgtgagt gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt
     1561 catcgctgag aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac
     1621 ctttgacaag ggcacgaaga ccggtggacc cttcggaacc atcaagcacc ctgccgaact
     1681 ggctcacagc gctaacaacg gtcttgacat cgctgttagg cttttggagc cactcaaggc
     1741 ggagttccct attttgagct acgccgattt ctaccagttg gctggcgttg ttgccgttga
     1801 ggtcacgggt ggacctaagg ttccattcca ccctggaaga gaggacaagc ctgagccacc
     1861 accagagggt cgcttgcccg atcccactaa gggttctgac catttgagag atgtgtttgg
     1921 caaagctatg gggcttactg accaagatat cgttgctcta tctgggggtc acactattgg
     1981 agctgcacac aaggagcgtt ctggatttga gggtccctgg acctctaatc ctcttatttt
     2041 cgacaactca tacttcacgg agttgttgag tggtgagaag gaaggtctcc ttcagctacc
     2101 ttctgacaag gctcttttgt ctgaccctgt attccgccct ctcgttgaca aatatgcagc
     2161 ggacgaagat gccttctttg ctgattacgc tgaggctcac caaaagcttt ccgagcttgg
     2221 gtttgctgat gccgtcgacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     2281 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     2341 tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     2401 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     2461 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     2521 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     2581 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     2641 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     2701 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     2761 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     2821 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     2881 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     2941 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     3001 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     3061 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     3121 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     3181 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     3241 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     3301 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     3361 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     3421 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3481 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3541 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3601 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3661 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3721 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3781 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3841 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3901 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3961 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4021 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4081 cctggccttt tgctggcctt ttgctcac
//
