LOCUS       sequence-205180-        4197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for 3' tagging with APEX2 (includes 
            cytoplasmic GFP reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4197)
  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 4197)
  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: p3E-APEX2-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..4197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..748)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             753..1499
                     /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             1509..1565
                     /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             join(1584..1586,1587..2300)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1584..1586,1587..2297)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1626..1647)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1887..1906)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2234..2255
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2306..2401)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2438..2457)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2439..2457)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2462..2478)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2462..2478)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2580..2599)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2591..3400
                     /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(2658..2677)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3547..4135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4036..4055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggtcg acggaaagtc ttacccaact gtgagtgctg
      781 attaccagga cgccgttgag aaggcgaaga agaagctcag aggcttcatc gctgagaaga
      841 gatgcgctcc tctaatgctc cgtttggcat tccactctgc tggaaccttt gacaagggca
      901 cgaagaccgg tggacccttc ggaaccatca agcaccctgc cgaactggct cacagcgcta
      961 acaacggtct tgacatcgct gttaggcttt tggagccact caaggcggag ttccctattt
     1021 tgagctacgc cgatttctac cagttggctg gcgttgttgc cgttgaggtc acgggtggac
     1081 ctaaggttcc attccaccct ggaagagagg acaagcctga gccaccacca gagggtcgct
     1141 tgcccgatcc cactaagggt tctgaccatt tgagagatgt gtttggcaaa gctatggggc
     1201 ttactgacca agatatcgtt gctctatctg ggggtcacac tattggagct gcacacaagg
     1261 agcgttctgg atttgagggt ccctggacct ctaatcctct tattttcgac aactcatact
     1321 tcacggagtt gttgagtggt gagaaggaag gtctccttca gctaccttct gacaaggctc
     1381 ttttgtctga ccctgtattc cgccctctcg ttgacaaata tgcagcggac gaagatgcct
     1441 tctttgctga ttacgctgag gctcaccaaa agctttccga gcttgggttt gctgatgccg
     1501 gatccggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg
     1561 gtcctgctcc tggaagcggt accgtgagca agggcgagga gctgttcacc ggggtggtgc
     1621 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1681 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1741 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1801 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1861 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1921 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1981 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2041 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2101 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2161 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2221 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2281 tggacgagct gtacaagtaa ctagtcaact ttattataca aagttggcat tataaaaaag
     2341 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     2401 tggagctcca tggtagcgtt aacgcggccg cgatatcccc tatagtgagt cgtattacat
     2461 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     2521 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     2581 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     2641 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     2701 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     2761 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     2821 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     2881 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     2941 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     3001 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     3061 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     3121 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     3181 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     3241 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     3301 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     3361 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     3421 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     3481 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3541 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3601 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3661 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3721 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3781 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3841 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3901 aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc
     3961 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4021 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4081 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4141 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
