LOCUS       sequence-220627-        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"
     protein_bind    complement(23..47)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             52..798
                     /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             808..864
                     /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(883..885,886..1599)
                     /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(883..885,886..1596)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(925..946)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1186..1205)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1533..1554
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1605..1700)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1737..1756)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1738..1756)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1761..1777)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1761..1777)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1879..1898)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1890..2699
                     /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(1957..1976)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2846..3434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3335..3354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3588..3605
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3764..3791)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3883..3969)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4018..4040
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4032..4049
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4033..4049
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tttatatcat tttacgtttc tcgttcaact ttcttgtaca aagtggtcga cggaaagtct
       61 tacccaactg tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga
      121 ggcttcatcg ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct
      181 ggaacctttg acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc
      241 gaactggctc acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc
      301 aaggcggagt tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc
      361 gttgaggtca cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag
      421 ccaccaccag agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg
      481 tttggcaaag ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact
      541 attggagctg cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt
      601 attttcgaca actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag
      661 ctaccttctg acaaggctct tttgtctgac cctgtattcc gccctctcgt tgacaaatat
      721 gcagcggacg aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag
      781 cttgggtttg ctgatgccgg atccggagcc acgaacttct ctctgttaaa gcaagcagga
      841 gacgtggaag aaaaccccgg tcctgctcct ggaagcggta ccgtgagcaa gggcgaggag
      901 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      961 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1021 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1081 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1141 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1201 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1261 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1321 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1381 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1441 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1501 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1561 gccgggatca ctctcggcat ggacgagctg tacaagtaac tagtcaactt tattatacaa
     1621 agttggcatt ataaaaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag
     1681 tcaaaataaa atcattattt ggagctccat ggtagcgtta acgcggccgc gatatcccct
     1741 atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa
     1801 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     1861 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     1921 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     1981 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     2041 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     2101 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2161 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     2221 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     2281 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     2341 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     2401 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     2461 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     2521 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     2581 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     2641 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     2701 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     2761 caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     2821 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2881 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2941 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3001 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3061 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3121 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3181 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3241 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3301 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3361 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3421 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3481 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3541 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3601 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3661 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3721 ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     3781 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     3841 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     3901 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     3961 ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg atgttttccc
     4021 agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg gccctgcagc tctagagctc
     4081 gaattctaca ggtcactaat accatctaag tagttggttc atagtgactg catatgttgt
     4141 gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat attgata
//
