LOCUS       sequence-117215-        6181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving APEX2-ML1N (Mouse 2xML1N domain 
            (mutant version), [marks PI(3,5)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 6181)
  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 6181)
  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-APEX2-2xML1N(7Q)
FEATURES             Location/Qualifiers
     source          1..6181
                     /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(2170..2190)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2230..2364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2254..2273)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2308..2327
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2477..2497)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2477..2495)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2516..2532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2516..2532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2529..2551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2540..2556
                     /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(2564..2570,2571..2588,2589..2594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2609..2630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2747..2764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2918..3506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2998..3017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3677..4468,4469..4537))
                     /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     4300..4319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4538..4642)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4668..5123)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4805..4826)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5017..5036
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5197..6181
                     /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 gtattctgat ctgactatgg ctacacctgc cgggagacgc
     1741 gcaagcgaaa ctgaaagact gctgacacca aaccctggct atggaaccca agtgggcaca
     1801 tctccagctc ccaccacacc cactgaggaa gaggacctgc agcagcagct gaagtacttc
     1861 tttatgagtc catgcgatca gttccaggca aaggggcagc agccctgtaa actgatgctg
     1921 agaatcctga caatggccac tcccgctggc aggagagcaa gcgagactga aaggctgctg
     1981 acccctaacc caggctacgg aacccaggtc ggaacatccc cagcacctac tacccctacc
     2041 gaagaggaag acctgcaaca acagctgaaa tatttcttta tgagcccttg cgatcagttt
     2101 caggccaagg ggcagcagcc atgtaaactg atgctggacc cccctgacct ggataattaa
     2161 ggatccgaac aactttatta tacatagttg cactcgagcc tctagaacta tagtgagtcg
     2221 tattacgtag atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     2281 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     2341 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     2401 cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg
     2461 tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     2521 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2581 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2641 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2701 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2761 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2821 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2881 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2941 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3001 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3061 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3121 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3181 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3241 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3301 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     3361 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3421 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3481 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3541 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3601 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3661 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3721 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3781 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3841 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3901 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3961 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4021 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4081 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4141 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4201 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4261 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4321 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4381 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4441 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4501 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4561 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4621 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     4681 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4741 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4801 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4861 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4921 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4981 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5041 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5101 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     5161 gcgatcggtg cgggcctctt cgctattacg ccagtcgacc atagccaatt caatatggcg
     5221 tatatggact catgccaatt caatatggtg gatctggacc tgtgccaatt caatatggcg
     5281 tatatggact cgtgccaatt caatatggtg gatctggacc ccagccaatt caatatggcg
     5341 gacttggcac catgccaatt caatatggcg gacttggcac tgtgccaact ggggaggggt
     5401 ctacttggca cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag tccgccatat
     5461 tgaattggca tggtgccaat aatggcggcc atattggcta tatgccagga tcaatatata
     5521 ggcaatatcc aatatggccc tatgccaata tggctattgg ccaggttcaa tactatgtat
     5581 tggccctatg ccatatagta ttccatatat gggttttcct attgacgtag atagcccctc
     5641 ccaatgggcg gtcccatata ccatatatgg ggcttcctaa taccgcccat agccactccc
     5701 ccattgacgt caatggtctc tatatatggt ctttcctatt gacgtcatat gggcggtcct
     5761 attgacgtat atggcgcctc ccccattgac gtcaattacg gtaaatggcc cgcctggctc
     5821 aatgcccatt gacgtcaata ggaccaccca ccattgacgt caatgggatg gctcattgcc
     5881 cattcatatc cgttctcacg ccccctattg acgtcaatga cggtaaatgg cccacttggc
     5941 agtacatcaa tatctattaa tagtaacttg gcaagtacat tactattgga aggacgccag
     6001 ggtacattgg cagtactccc attgacgtca atggcggtaa atggcccgcg atggctgcca
     6061 agtacatccc cattgacgtc aatggggagg ggcaatgacg caaatgggcg ttccattgac
     6121 gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc gtttagggaa
     6181 c
//
