LOCUS       sequence-117187-        6082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving APEX2-TAPP1 (Human TAPP1-PH 
            domain, [marks PI(3,4)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 6082)
  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 6082)
  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: mKate2-P2A-APEX2-TAPP1-PH
FEATURES             Location/Qualifiers
     source          1..6082
                     /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      133..142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             139..834
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             850..906
                     /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             925..1671
                     /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(1676..1700)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2071..2091)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2131..2265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2155..2174)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2209..2228
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2378..2398)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2378..2396)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2417..2433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2417..2433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2430..2452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2441..2457
                     /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(2465..2471,2472..2489,2490..2495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2510..2531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2648..2665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2819..3407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2899..2918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3578..4369,4370..4438))
                     /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     4201..4220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4439..4543)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4569..5024)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4706..4727)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4918..4937
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5098..6082
                     /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 ctgccaccat ggtgagcgag ctgattaagg agaacatgca catgaagctg
      181 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
      241 ccctacgagg gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc
      301 gccttcgaca tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc
      361 cagggcatcc ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc
      421 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
      481 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
      541 cagaagaaaa cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc
      601 ctggaaggca gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
      661 ttgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
      721 tatgtggaca gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
      781 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca cagacctagg
      841 ggcagcggcg ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc
      901 ggccccgccc ccggcagcct cgagggaaag tcttacccaa ctgtgagtgc tgattaccag
      961 gacgccgttg agaaggcgaa gaagaagctc agaggcttca tcgctgagaa gagatgcgct
     1021 cctctaatgc tccgtttggc attccactct gctggaacct ttgacaaggg cacgaagacc
     1081 ggtggaccct tcggaaccat caagcaccct gccgaactgg ctcacagcgc taacaacggt
     1141 cttgacatcg ctgttaggct tttggagcca ctcaaggcgg agttccctat tttgagctac
     1201 gccgatttct accagttggc tggcgttgtt gccgttgagg tcacgggtgg acctaaggtt
     1261 ccattccacc ctggaagaga ggacaagcct gagccaccac cagagggtcg cttgcccgat
     1321 cccactaagg gttctgacca tttgagagat gtgtttggca aagctatggg gcttactgac
     1381 caagatatcg ttgctctatc tgggggtcac actattggag ctgcacacaa ggagcgttct
     1441 ggatttgagg gtccctggac ctctaatcct cttattttcg acaactcata cttcacggag
     1501 ttgttgagtg gtgagaagga aggtctcctt cagctacctt ctgacaaggc tcttttgtct
     1561 gaccctgtat tccgccctct cgttgacaaa tatgcagcgg acgaagatgc cttctttgct
     1621 gattacgctg aggctcacca aaagctttcc gagcttgggt ttgctgatgc cgtcgaccca
     1681 gctttcttgt acaaagtggt gtttactcct aaaccacctc aagatagtgc ggttatcaaa
     1741 gctggatatt gtgtaaaaca aggagcagtg atgaaaaact ggaagagaag atattttcaa
     1801 ttggatgaaa acacaatagg ctacttcaaa tctgaactgg aaaaggaacc tcttcgcgta
     1861 ataccactta aagaggttca taaagtccag gaatgtaagc aaagcgacat aatgatgagg
     1921 gacaacctct ttgaaattgt aacaacgtct cgaactttct atgtgcaggc tgatagccct
     1981 gaagagatgc acagttggat taaagcagtc tctggcgcca ttgtagcaca gcggggtccc
     2041 ggcagatctg cgtcttctga gcatccctga caactttatt atacatagtt gcactcgagc
     2101 ctctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     2161 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2221 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2281 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg
     2341 gcgccaatgc attgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc
     2401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2461 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3241 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3421 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4561 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4621 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4681 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4741 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4801 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4861 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4921 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     4981 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag
     5041 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagtcgac
     5101 catagccaat tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac
     5161 ctgtgccaat tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac
     5221 cccagccaat tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacttggca
     5281 ctgtgccaac tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc
     5341 cctgtgccaa gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct
     5401 atatgccagg atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg
     5461 gccaggttca atactatgta ttggccctat gccatatagt attccatata tgggttttcc
     5521 tattgacgta gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta
     5581 ataccgccca tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat
     5641 tgacgtcata tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac
     5701 ggtaaatggc ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg
     5761 tcaatgggat ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg
     5821 acggtaaatg gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca
     5881 ttactattgg aaggacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta
     5941 aatggcccgc gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac
     6001 gcaaatgggc gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat
     6061 aagcaatgct cgtttaggga ac
//
