LOCUS       sequence-116993-        6243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving BTK-APEX2 (Human BTK-PH domain, 
            [marks PI(3,4,5)P3] fused to EM peroxidase marker). Also contains 
            bicistronially expressed cytoplasmic red fluorescent reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6243)
  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 6243)
  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: BTK-APEX2-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..6243
                     /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"
     protein_bind    complement(671..695)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             700..1446
                     /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             1456..1512
                     /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             1531..2226
                     /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(2232..2252)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2292..2426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2316..2335)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2370..2389
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2539..2559)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2539..2557)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2578..2594)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2578..2594)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2591..2613)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2602..2618
                     /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(2626..2632,2633..2650,2651..2656))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2671..2692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2809..2826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2980..3568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3060..3079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3739..4530,4531..4599))
                     /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     4362..4381
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4600..4704)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4730..5185)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4867..4888)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5079..5098
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5259..6243
                     /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 ggccgcagtg attctggaga gcatctttct gaagcgatcc
      181 caacagaaaa agaaaacatc acctctaaac ttcaagaagc gcctgtttct cttgaccgtg
      241 cacaaactct cctactatga gtatgacttt gaacgtggga gaagaggcag taagaagggt
      301 tcaatagatg ttgagaagat cacttgtgtt gaaacagtgg ttcctgaaaa aaatcctcct
      361 ccagaaagac agattccgag aagaggtgaa gagtccagtg aaatggagca aatttcaatc
      421 attgaaaggt tcccttatcc cttccaggtt gtatatgatg aagggcctct ctacgtcttc
      481 tccccaactg aagaactaag gaagcggtgg attcaccagc tcaaaaacgt aatccggtac
      541 aacagtgatc tggttcagaa atatcaccct tgcttctgga tcgatgggca gtatctctgc
      601 tgctctcaga cagccaaaaa tgctatgggc tgccaaattt tggagaacag gaatggaagc
      661 ttaaaaccgg acccagcttt cttgtacaaa gtggtcgacg gaaagtctta cccaactgtg
      721 agtgctgatt accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct
      781 gagaagagat gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac
      841 aagggcacga agaccggtgg acccttcgga accatcaagc accctgccga actggctcac
      901 agcgctaaca acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc
      961 cctattttga gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg
     1021 ggtggaccta aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag
     1081 ggtcgcttgc ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct
     1141 atggggctta ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca
     1201 cacaaggagc gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac
     1261 tcatacttca cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac
     1321 aaggctcttt tgtctgaccc tgtattccgc cctctcgttg acaaatatgc agcggacgaa
     1381 gatgccttct ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct
     1441 gatgccggat ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
     1501 aaccccggtc ctgctcctgg aagcggtacc gtgagcgagc tgattaagga gaacatgcac
     1561 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
     1621 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggcggtcga gggcggccct
     1681 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc
     1741 aaccacaccc agggcatccc cgacttcttt aagcagtcct tccccgaggg cttcacatgg
     1801 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     1861 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc
     1921 cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagaccct gtaccccgct
     1981 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg
     2041 atctgcaact tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc
     2101 ggcgtctact atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac
     2161 gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac
     2221 agatgactag tcaactttat tatacatagt tgcactcgag cctctagaac tatagtgagt
     2281 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     2341 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2401 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     2461 ttcaggggga ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc
     2521 ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2581 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2641 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2701 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2761 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2821 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2881 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2941 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3001 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3061 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3121 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3181 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3241 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3301 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3361 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3421 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3481 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3541 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3601 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3661 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3721 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3781 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3841 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3901 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3961 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4021 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4081 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4141 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4201 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4261 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4321 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4381 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4441 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4501 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4561 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4621 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4681 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     4741 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4801 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     4861 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4921 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4981 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5041 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5101 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5161 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     5221 gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga ccatagccaa ttcaatatgg
     5281 cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa ttcaatatgg
     5341 cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa ttcaatatgg
     5401 cggacttggc accatgccaa ttcaatatgg cggacttggc actgtgccaa ctggggaggg
     5461 gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca agtccgccat
     5521 attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag gatcaatata
     5581 taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc aatactatgt
     5641 attggcccta tgccatatag tattccatat atgggttttc ctattgacgt agatagcccc
     5701 tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc atagccactc
     5761 ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat atgggcggtc
     5821 ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg cccgcctggc
     5881 tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga tggctcattg
     5941 cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat ggcccacttg
     6001 gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg gaaggacgcc
     6061 agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg cgatggctgc
     6121 caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg cgttccattg
     6181 acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc tcgtttaggg
     6241 aac
//
