LOCUS       sequence-271567-        5633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a protein of interest fused to the C-terminus of 
            Flag-2xAPEX2 for proximity labeling or TEM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5633)
  AUTHORS   Ken-Ichi Takemaru
  TITLE     2xAPEX2
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5633)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Flag-2xAPEX2-C Cloning Vector
FEATURES             Location/Qualifiers
     source          1..5633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        845..863
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     845..862
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             901..924
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             931..1677
                     /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             1681..2424
                     /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="KSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTF
                     DKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVE
                     VTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTI
                     GAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKY
                     AADEDAFFADYAEAHQKLSELGFADA"
     polyA_signal    2495..2629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2519..2538)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2573..2592
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2742..2762)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2742..2760)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2781..2797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2781..2797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2794..2816)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2805..2821
                     /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(2829..2835,2836..2853,2854..2859))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2874..2895
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3012..3029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3183..3771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3263..3282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3942..4733,4734..4802))
                     /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     4565..4584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4803..4907)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4933..5388)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5070..5091)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5282..5301
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5459..5633,1..810)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 acctggcact gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg
       61 gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca
      121 tattggctat atgccaggat caatatatag gcaatatcca atatggccct atgccaatat
      181 ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg
      241 ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg
      301 gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc
      361 tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg
      421 tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac
      481 cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga
      541 cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg
      601 caagtacatt actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa
      661 tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg
      721 gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag
      781 gtctatataa gcaatgctcg tttagggaac cgccattctg cctggggacg tcggagcaag
      841 cttgatttag gtgacactat agaatacaag ctacttgttc tttttgcagg atccaccatg
      901 gattacaagg atgacgacga taagatccgc ggaaagtctt acccaactgt gagtgctgat
      961 taccaggacg ccgttgagaa ggcgaagaag aagctcagag gcttcatcgc tgagaagaga
     1021 tgcgctcctc taatgctccg tttggcattc cactctgctg gaacctttga caagggcacg
     1081 aagaccggtg gacccttcgg aaccatcaag caccctgccg aactggctca cagcgctaac
     1141 aacggtcttg acatcgctgt taggcttttg gagccactca aggcggagtt ccctattttg
     1201 agctacgccg atttctacca gttggctggc gttgttgccg ttgaggtcac gggtggacct
     1261 aaggttccat tccaccctgg aagagaggac aagcctgagc caccaccaga gggtcgcttg
     1321 cccgatccca ctaagggttc tgaccatttg agagatgtgt ttggcaaagc tatggggctt
     1381 actgaccaag atatcgttgc tctatctggg ggtcacacta ttggagctgc acacaaggag
     1441 cgttctggat ttgagggtcc ctggacctct aatcctctta ttttcgacaa ctcatacttc
     1501 acggagttgt tgagtggtga gaaggaaggt ctccttcagc taccttctga caaggctctt
     1561 ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg cagcggacga agatgccttc
     1621 tttgctgatt acgctgaggc tcaccaaaag ctttccgagc ttgggtttgc tgatgccgta
     1681 aagtcttacc caactgtgag tgctgattac caggacgccg ttgagaaggc gaagaagaag
     1741 ctcagaggct tcatcgctga gaagagatgc gctcctctaa tgctccgttt ggcattccac
     1801 tctgctggaa cctttgacaa gggcacgaag accggtggac ccttcggaac catcaagcac
     1861 cctgccgaac tggctcacag cgctaacaac ggtcttgaca tcgctgttag gcttttggag
     1921 ccactcaagg cggagttccc tattttgagc tacgccgatt tctaccagtt ggctggcgtt
     1981 gttgccgttg aggtcacggg tggacctaag gttccattcc accctggaag agaggacaag
     2041 cctgagccac caccagaggg tcgcttgccc gatcccacta agggttctga ccatttgaga
     2101 gatgtgtttg gcaaagctat ggggcttact gaccaagata tcgttgctct atctgggggt
     2161 cacactattg gagctgcaca caaggagcgt tctggatttg agggtccctg gacctctaat
     2221 cctcttattt tcgacaactc atacttcacg gagttgttga gtggtgagaa ggaaggtctc
     2281 cttcagctac cttctgacaa ggctcttttg tctgaccctg tattccgccc tctcgttgac
     2341 aaatatgcag cggacgaaga tgccttcttt gctgattacg ctgaggctca ccaaaagctt
     2401 tccgagcttg ggtttgctga tgccgtcgac ggatctcaga tctcgaattc aaggcctctc
     2461 gagcctctag aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga
     2521 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2581 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     2641 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc
     2701 cgcggcgcca atgcattggg cccggtaccc agcttttgtt ccctttagtg agggttaatt
     2761 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2821 attccacaca acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg
     2881 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2941 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3001 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3061 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4921 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     4981 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5041 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5101 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5161 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5221 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5281 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5341 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     5401 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt
     5461 cgaccgccaa ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga
     5521 cctgtgccaa ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga
     5581 ccccagccaa ttcaatatgg cggacttggc accatgccaa ttcaatatgg cgg
//
