LOCUS       sequence-127760-        6627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving 2PH-PLC-delta-APEX2 (Human 
            2xPLCd PH domain, [marks PI(4,5)P2] fused to EM peroxidase marker). 
            Also contains bicistronially expressed cytoplasmic red reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6627)
  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 6627)
  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: 2PH-PLCd-APEX2-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..6627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      136..145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1055..1079)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1084..1830
                     /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             1840..1896
                     /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             1915..2610
                     /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(2616..2636)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2676..2810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2700..2719)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2754..2773
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2923..2943)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2923..2941)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2962..2978)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2962..2978)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2975..2997)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2986..3002
                     /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(3010..3016,3017..3034,3035..3040))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3055..3076
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3193..3210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3364..3952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3444..3463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4123..4914,4915..4983))
                     /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     4746..4765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4984..5088)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5114..5569)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5251..5272)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5463..5482
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5643..6627
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccgccac catggactcg ggccgggact tcctgaccct gcacggccta
      181 caggatgatg aggatctaca ggcgctgctg aagggcagcc agctcctgaa ggtgaagtcc
      241 agctcatgga ggagagagcg cttctacaag ttgcaggagg actgcaagac catctggcag
      301 gagtcccgca aggtcatgcg gaccccggag tcccagctgt tctccatcga ggacattcag
      361 gaggtgcgaa tggggcaccg cacggagggt ctggagaagt tcgcccgtga tgtgcccgag
      421 gaccgctgct tctccattgt cttcaaggac cagcgcaata cactagacct catcgcccca
      481 tcgccagctg atgcccagca ctgggtgctg gggctgcaca aaattatcca tcacagcgga
      541 tctatggacc agcgtcagaa gctacagcac tggattcact cctgcttgcg aaaagctgac
      601 aaaaacaagg acaacaagat aggcggcagc ggcggcagcg gcggcgatag cggtagagat
      661 tttctgactc tgcatggact gcaggacgac gaggacctgc aggccctgct gaagggcagc
      721 cagctgctga aggtgaagag cagcagctgg cgccgcgagc gcttctacaa gctgcaggag
      781 gactgcaaga ccatctggca ggagagccgc aaggtgatgc gcacccccga gagccagctg
      841 ttcagcatcg aggacatcca ggaggtgcgc atgggccacc gcaccgaggg cctggagaag
      901 ttcgcccgcg acgtgcccga ggaccgctgc ttcagcatcg tgttcaagga ccagcgcaac
      961 accctggacc tgatcgcccc cagccccgcc gacgcccagc actgggtgct gggcctgcac
     1021 aagatcattc accattctgg cagcatggat gggtacccag ctttcttgta caaagtggtc
     1081 gacggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
     1141 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
     1201 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
     1261 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
     1321 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
     1381 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
     1441 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat
     1501 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
     1561 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
     1621 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
     1681 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
     1741 gttgacaaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
     1801 aagctttccg agcttgggtt tgctgatgcc ggatccggag ccacgaactt ctctctgtta
     1861 aagcaagcag gagacgtgga agaaaacccc ggtcctgctc ctggaagcgg taccgtgagc
     1921 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac
     1981 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     2041 atcaaggcgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc
     2101 atgtacggca gcaaaacctt catcaaccac acccagggca tccccgactt ctttaagcag
     2161 tccttccccg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     2221 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     2281 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     2341 tccaccgaga ccctgtaccc cgctgacggc ggcctggaag gcagagccga catggccctg
     2401 aagctcgtgg gcgggggcca cctgatctgc aacttgaaga ccacatacag atccaagaaa
     2461 cccgctaaga acctcaagat gcccggcgtc tactatgtgg acagaagact ggaaagaatc
     2521 aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc
     2581 gacctcccta gcaaactggg gcacagatga ctagtcaact ttattataca tagttgcact
     2641 cgagcctcta gaactatagt gagtcgtatt acgtagatcc agacatgata agatacattg
     2701 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     2761 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     2821 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taattcgcgg
     2881 ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt tccctttagt gagggttaat
     2941 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3001 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3061 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3121 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3301 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3361 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3421 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3481 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3541 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3601 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3661 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3721 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3781 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3841 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3901 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3961 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4021 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4081 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4141 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4201 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4261 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4321 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4381 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4441 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4501 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4561 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4621 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4681 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4741 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4801 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4861 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4921 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4981 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5041 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5101 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5161 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5221 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5281 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5341 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5401 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5461 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5521 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     5581 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     5641 tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc
     5701 tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc
     5761 tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact
     5821 tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc
     5881 ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat
     5941 tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc
     6001 tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt
     6061 tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct
     6121 tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt
     6181 cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca
     6241 attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat
     6301 tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt
     6361 caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa
     6421 gtacattact attggaagga cgccagggta cattggcagt actcccattg acgtcaatgg
     6481 cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca
     6541 atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc
     6601 tatataagca atgctcgttt agggaac
//
