LOCUS       sequence-117189-        6013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving APEX2-OSBP (Human OSBP domain, 
            [marks PI4P] 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 6013)
  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 6013)
  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-OSBP
FEATURES             Location/Qualifiers
     source          1..6013
                     /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(2002..2022)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2062..2196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2086..2105)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2140..2159
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2309..2329)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2309..2327)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2348..2364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2348..2364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2361..2383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2372..2388
                     /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(2396..2402,2403..2420,2421..2426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2441..2462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2579..2596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2750..3338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2830..2849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3509..4300,4301..4369))
                     /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     4132..4151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4370..4474)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4500..4955)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4637..4658)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4849..4868
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5029..6013
                     /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 acaaagtggc ttcgggctcg gctcgagagg gctggctctt caaatggacc
     1741 aattatatca aaggctacca gcggcgatgg ttcgtgctga gcaacgggct cctgagctac
     1801 tacagatcaa aggcagagat gagacatacc tgccgtggta ccatcaacct cgccacagcc
     1861 aacatcaccg tggaggactc ctgcaacttc atcatttcca atgggggtgc tcagacctac
     1921 catctgaaag ctagttcaga agttgagcgg cagcgctggg tgacggccct ggaactggcc
     1981 aaggccaaag ctgtgaagtg acaactttat tatacatagt tgcactcgag cctctagaac
     2041 tatagtgagt cgtattacgt agatccagac atgataagat acattgatga gtttggacaa
     2101 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2161 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     2221 atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg
     2281 cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     2341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3181 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     4501 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     4561 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     4621 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     4681 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     4741 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     4801 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     4861 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     4921 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     4981 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga ccatagccaa
     5041 ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa
     5101 ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa
     5161 ttcaatatgg cggacttggc accatgccaa ttcaatatgg cggacttggc actgtgccaa
     5221 ctggggaggg gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca
     5281 agtccgccat attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag
     5341 gatcaatata taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc
     5401 aatactatgt attggcccta tgccatatag tattccatat atgggttttc ctattgacgt
     5461 agatagcccc tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc
     5521 atagccactc ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat
     5581 atgggcggtc ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg
     5641 cccgcctggc tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga
     5701 tggctcattg cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat
     5761 ggcccacttg gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg
     5821 gaaggacgcc agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg
     5881 cgatggctgc caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg
     5941 cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc
     6001 tcgtttaggg aac
//
