LOCUS       sequence-117209-        4176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway entry clone for adding C-terminal fusions of 
            APEX2-P2A-mKate2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4176)
  AUTHORS   Ariotti N, Hall TE, Rae J, Ferguson C, McMahon KA, Martel N, Webb 
            RE, Webb RI, Teasdale RD, Parton RG
  TITLE     Modular Detection of GFP-Labeled Proteins for Rapid Screening by 
            Electron Microscopy in Cells and Organisms.
  JOURNAL   Dev Cell. 2015 Nov 23;35(4):513-25. doi: 
            10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.
  PUBMED    26585296
REFERENCE   2  (bases 1 to 4176)
  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"; doi: 
            "10.1016/j.devcel.2015.10.016"; journalName: "Dev Cell"; date: 
            "2015-11-23- 23"; volume: "35"; issue: "4"; pages: "513-25"
COMMENT     Sequence Label: p3E-APEX2-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..4176
                     /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             1584..2279
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     protein_bind    complement(2285..2380)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2417..2436)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2418..2436)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2441..2457)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2441..2457)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2559..2578)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2570..3379
                     /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(2637..2656)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3526..4114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4015..4034
                     /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 accgtgagcg agctgattaa ggagaacatg cacatgaagc
     1621 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     1681 agccctacga gggcacccag accatgagaa tcaaggcggt cgagggcggc cctctcccct
     1741 tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca
     1801 cccagggcat ccccgacttc tttaagcagt ccttccccga gggcttcaca tgggagagag
     1861 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     1921 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     1981 tgcagaagaa aacactcggc tgggaggcct ccaccgagac cctgtacccc gctgacggcg
     2041 gcctggaagg cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca
     2101 acttgaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     2161 actatgtgga cagaagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     2221 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacagatgac
     2281 tagtcaactt tattatacaa agttggcatt ataaaaaagc attgcttatc aatttgttgc
     2341 aacgaacagg tcactatcag tcaaaataaa atcattattt ggagctccat ggtagcgtta
     2401 acgcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca
     2461 gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     2521 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     2581 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     2641 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa
     2701 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     2761 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     2821 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
     2881 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     2941 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     3001 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     3061 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     3121 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     3181 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     3241 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     3301 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     3361 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     3421 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc
     3481 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3541 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3601 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3661 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3721 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3781 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3841 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3901 atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3961 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4021 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4081 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4141 gttcctggcc ttttgctggc cttttgctca catgtt
//
