LOCUS       sequence-213263-        5226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Theophylline-inducible expression of periplasmic APEX2 from a 
            mycobacterial shuttle plasmid (episomal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5226)
  AUTHORS   Ganapathy US, Bai L, Wei L, Eckartt KA, Lett CM, Previti ML, Carrico
            IS, Seeliger JC
  TITLE     Compartment-Specific Labeling of Bacterial Periplasmic Proteins by 
            Peroxidase-Mediated Biotinylation.
  JOURNAL   ACS Infect Dis. 2018 Apr 30. doi: 10.1021/acsinfecdis.8b00044.
  PUBMED    29708735
REFERENCE   2  (bases 1 to 5226)
  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: "ACS Infect 
            Dis. 2018 Apr 30. doi: 10.1021/acsinfecdis.8b00044."
COMMENT     Sequence Label: pRibo-Sec-APEX2
FEATURES             Location/Qualifiers
     source          1..5226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      339..348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             389..430
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             431..1177
                     /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"
     terminator      1247..1293
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1439..1458)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1450..2265
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1523..1542)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2597..3185
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3086..3105
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctagaggtg accacaacgc gcccgctttg atcggggacg tctgcggccg accatttacg
       61 ggtcttgttg tcgttggcgg tcatgggccg aacatactca cccggatcgg agggccgagg
      121 acaaggtcga acgaggggca tgacccggtg cggggcttct tgcactcggc ataggcgagt
      181 gctaagaata acgttggcac tcgcgaccgg gatacgactc actataggta ccggtgatac
      241 cagcatcgtc ttgatgccct tggcagcacc ctgctaagga ggcaacaaga tgcatatgaa
      301 gctcaccaca atgatcaaga cggcagtagc ggtcgtggcc atggcggcca tcgcgacctt
      361 tgcggcaccg gtcgcgttgg ctggatccgg caagcccatc cccaaccccc tgctgggcct
      421 ggacagcacc ggaaagtctt acccaactgt gagtgctgat taccaggacg ccgttgagaa
      481 ggcgaagaag aagctcagag gcttcatcgc tgagaagaga tgcgctcctc taatgctccg
      541 tttggcattc cactctgctg gaacctttga caagggcacg aagaccggtg gacccttcgg
      601 aaccatcaag caccctgccg aactggctca cagcgctaac aacggtcttg acatcgctgt
      661 taggcttttg gagccactca aggcggagtt ccctattttg agctacgccg atttctacca
      721 gttggctggc gttgttgccg ttgaggtcac gggtggacct aaggttccat tccaccctgg
      781 aagagaggac aagcctgagc caccaccaga gggtcgcttg cccgatccca ctaagggttc
      841 tgaccatttg agagatgtgt ttggcaaagc tatggggctt actgaccaag atatcgttgc
      901 tctatctggg ggtcacacta ttggagctgc acacaaggag cgttctggat ttgagggtcc
      961 ctggacctct aatcctctta ttttcgacaa ctcatacttc acggagttgt tgagtggtga
     1021 gaaggaaggt ctccttcagc taccttctga caaggctctt ttgtctgacc ctgtattccg
     1081 ccctctcgtt gataaatatg cagcggacga agatgccttc tttgctgatt acgctgaggc
     1141 tcaccaaaag ctttccgagc ttgggtttgc tgatgcctaa tgaagatgga gaccaagctt
     1201 atcgatgtcg acgtagttaa ctagcgtacg atcgactgcc aggcatcaaa taaaacgaaa
     1261 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtccggcc atcatggccg
     1321 cggtgatcag ctagccaaca aagcgacgtt gtgtctcaaa atctctgatg ttacattgca
     1381 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     1441 aggggtgtta tgagccatat tcaacgggaa acgtcttgct cgaggccgcg attaaattcc
     1501 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     1561 gcgacaatct atcgcttgta tgggaagccc catgcgccag agttgtttct gaaacatggc
     1621 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     1681 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     1741 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     1801 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     1861 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     1921 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     1981 gtctggaaag aaatgcataa tcttttgcca ttctcaccgg attcagtcgt cactcatggt
     2041 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     2101 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     2161 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     2221 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     2281 tggttgtaac actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa
     2341 atcgaacttt tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt
     2401 ccgtggcaaa gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca
     2461 accgtggctc cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag
     2521 caacaccttc ttcacgaggc agacctcact agttccactg agcgtcagac cccgtagaaa
     2581 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2641 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2701 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2761 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2821 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2881 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2941 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg
     3001 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3061 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3121 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3181 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3241 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3301 gagctgatac cgctcgccgc agccgaacga ccgagcgcaa cgcgtgagcc caccagctcc
     3361 gtaagttcgg gtgctgtgtg gctcgtaccc gcgcattcag gcggcagggg gtctaacggg
     3421 tctaaggcgg cgtgtacggc cgccacagcg gctcttagcg gcccggaaac gtcctcgaaa
     3481 cgacgcatgt gttcctcctg gttggtacag gtggttgggg gtgctcggct gtcgctggtg
     3541 tttcatcatc agggctcgac gggagagcgg gggagtgtgc agttgtgggg tggcccctca
     3601 gcgaaatatc tgacttggag ctcgtgtcgg accatacacc ggtgattaat cgtggtttat
     3661 tatcaagcgt gagccacgtc gccgacgaat ttgagcagct ctggctgccg tactggtccc
     3721 tggcaagcga cgatctgctc gaggggatct accgccaaag ccgcgcgtcg gccctaggcc
     3781 gccggtacat cgaggcgaac ccaacagcgc tggcaaacct gctggtcgtg gacgtagacc
     3841 atccagacgc agcgctccga gcgctcagcg cccgggggtc ccatccgctg cccaacgcga
     3901 tcgtgggcaa tcgcgccaac ggccacgcac acgcagtgtg ggcactcaac gcccctgttc
     3961 cacgcaccga atacgcgcgg cgtaagccgc gccgtcgatg gcgaccgcag ttactcaggc
     4021 ctcatgacca aaaaccccgg ccacatcgcc tgggaaacgg aatggctcca ctcagatctc
     4081 tacacactca gccacatcga ggccgagctc ggcgcgaaca tgccaccgcc gcgctggcgt
     4141 cagcagacca cgtacaaagc ggctccgacg ccgctagggc ggaattgcgc actgttcgat
     4201 tccgtcaggt tgtgggccta tcttcccgcc ctcatgcgga tctacctgcc gacccggaac
     4261 gtggacggac tcggccgcgc gatctatgcc gagtgccacg cgcgaaacgc cgaatttccg
     4321 tgcaacgacg tgtgtcccgg accgctaccg gacagcgagg tccgcgccat cgccaacagc
     4381 atttggcgtt ggatcacaac caagtcgcgc atttgggcgg acgggatcgt ggtctacgag
     4441 gccacactca gtgcgcgcca tgcggccatc tcgcggaagg gcgcagcagc gcgcacggcg
     4501 gcgagcacag ttgcgcggcg cgcaaagtcc gcgtcagcca tggaggcatt gctatgagcg
     4561 acggctacag cgacggctac agcgacggct acaactggca gccgactgtc cgcaaaaagc
     4621 ggcgcgtgac cgccgccgaa ggcgctcgaa tcaccggact atccgaacgc cacgtcgtcc
     4681 ggctcgtggc gcaggaacgc agcgagtggt tcgccgagca ggctgcacgc cgcgaacgca
     4741 tccgcgccta tcacgacgac gagggccact cttggccgca aacggccaaa catttcgggc
     4801 tgcatctgga caccgttaag cgactcggct atcgggcgag gaaagagcgt gcggcagaac
     4861 aggaagcggc tcaaaaggcc cacaacgaag ccgacaatcc accgctgttc taacgcaatt
     4921 ggggagcggg tgtcgcgggg gttccgtggg gggttccgtt gcaacgggtc ggacaggtaa
     4981 aagtcctggt agacgctagt tttctggttt gggccatgcc tgtctcgttg cgtgtttcgt
     5041 tgcgtccgtt ttgaatacca gccagacgag acggggttct acgaatcttg gtcgatacca
     5101 agccatttcc gctgaatatc gtggagctca ccgccagaat cggtggttgt ggtgatgtac
     5161 gtggcgaact ccgttgtagt gcttgtggtg gcatccgtgg cgcggccgcg gtaccagatc
     5221 tttaaa
//
