LOCUS       sequence-221257-        5255 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 5255)
  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 5255)
  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..5255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..42
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             43..789
                     /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      859..905
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1055..1074)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1066..1881
                     /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(1139..1158)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2212..2800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2701..2720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      5206..5215
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ggcaagccca tccccaaccc cctgctgggc ctggacagca ccggaaagtc ttacccaact
       61 gtgagtgctg attaccagga cgccgttgag aaggcgaaga agaagctcag aggcttcatc
      121 gctgagaaga gatgcgctcc tctaatgctc cgtttggcat tccactctgc tggaaccttt
      181 gacaagggca cgaagaccgg tggacccttc ggaaccatca agcaccctgc cgaactggct
      241 cacagcgcta acaacggtct tgacatcgct gttaggcttt tggagccact caaggcggag
      301 ttccctattt tgagctacgc cgatttctac cagttggctg gcgttgttgc cgttgaggtc
      361 acgggtggac ctaaggttcc attccaccct ggaagagagg acaagcctga gccaccacca
      421 gagggtcgct tgcccgatcc cactaagggt tctgaccatt tgagagatgt gtttggcaaa
      481 gctatggggc ttactgacca agatatcgtt gctctatctg ggggtcacac tattggagct
      541 gcacacaagg agcgttctgg atttgagggt ccctggacct ctaatcctct tattttcgac
      601 aactcatact tcacggagtt gttgagtggt gagaaggaag gtctccttca gctaccttct
      661 gacaaggctc ttttgtctga ccctgtattc cgccctctcg ttgataaata tgcagcggac
      721 gaagatgcct tctttgctga ttacgctgag gctcaccaaa agctttccga gcttgggttt
      781 gctgatgcct aatgaagatg gagaccaagc ttatcgatgt cgacgtagtt aactagcgta
      841 cgatcgactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
      901 tttatgccat catggccgcg gtgatcagct agccacctga cgtcgggggg gggggaaagc
      961 cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     1021 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     1081 cgggaaacgt cttgctcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     1141 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     1201 aagccccatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     1261 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     1321 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     1381 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     1441 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     1501 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     1561 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataatctt
     1621 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     1681 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     1741 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     1801 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     1861 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     1921 acgctgactt gacgggacgg cggctttgtt gaataaatcg aacttttgct gagttgaagg
     1981 atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa
     2041 tcaccaactg gtccacctac aacaaagctc tcaccaaccg tggctccctc actttctggc
     2101 tggatgatgg ggcgattcag gcctggtatg agtcagcaac accttcttca cgaggcagac
     2161 ctcactagtt ccatgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2221 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2281 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2341 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2401 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2461 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2521 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2581 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgagg ggagaaaggc
     2641 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2701 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2761 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2821 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2881 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2941 aacgaccgag cgcaacgcgt gagcccacca gctccgtaag ttcgggcgct gtgtggctcg
     3001 tacccgcgca ttcaggcggc agggggtcta acgggtctaa ggcggcgtgt acggccgcca
     3061 cagcggctct cagcggcccg gaaacgtcct cgaaacgacg catgtgttcc tcctggttgg
     3121 tacaggtggt tgggggtgct cggctgtcgc tggtgttcca ccaccagggc tcgacgggag
     3181 agcgggggag tgtgcagttg tggggtggcc cctcagcgaa atatctgact tggagctcgt
     3241 gtcggaccat acaccggtga ttaatcgtgg tctactacca agcgtgagcc acgtcgccga
     3301 cgaatttgag cagctctggc tgccgtactg gccgctggca agcgacgatc tgctcgaggg
     3361 gatctaccgc caaagccgcg cgtcggccct aggccgccgg tacatcgagg cgaacccaac
     3421 agcgctggca aacctgctgg tcgtggacgt agaccatcca gacgcagcgc tccgagcgct
     3481 cagcgcccgg gggtcccatc cgctgcccaa cgcgatcgtg ggcaatcgcg ccaacggcca
     3541 cgcacacgca gtgtgggcac tcaacgcccc tgttccacgc accgaatacg cgcggcgtaa
     3601 gccgctcgca tacatggcgg cgtgcgccga aggccttcgg cgcgccgtcg atggcgaccg
     3661 cagttactca ggcctcatga ccaaaaaccc cggccacatc gcctgggaaa cggaatggct
     3721 ccactcagat ctctacacac tcagccacat cgaggccgag ctcggcgcga acatgccacc
     3781 gccgcgctgg cgtcagcaga ccacgtacaa agcggctccg acgccgctag ggcggaattg
     3841 cgcactgttc gattccgtca ggttgtgggc ctatcgtccc gccctcatgc ggatctacct
     3901 gccgacccgg aacgtggacg gactcggccg cgcgatctat gccgagtgcc acgcgcgaaa
     3961 cgccgaattt ccgtgcaacg acgtgtgtcc cggaccgcta ccggacagcg aggtccgcgc
     4021 catcgccaac agcatttggc gttggatcac aaccaagtcg cgcatttggg cggacgggat
     4081 cgtggtctac gaggccacac tcagtgcgcg ccagtcggcc atctcgcgga agggcgcagc
     4141 agcgcgcacg gcggcgagca cagttgcgcg gcgcgcaaag tccgcgtcag ccatggaggc
     4201 attgctatga gcgacggcta cagcgacggc tacaaccggc agccgactgt ccgcaaaaag
     4261 cggcgcgtga ccgccgccga aggcgctcga atcaccggac tatccgaacg ccacgtcgtc
     4321 cggctcgtgg cgcaggaacg cagcgagtgg ctcgccgagc aggctgcacg ccgcgaacgc
     4381 atccgcgcct atcacgacga cgagggccac tcttggccgc aaacggccaa acatttcggg
     4441 ctgcatctgg acaccgttaa gcgactcggc tatcgggcga ggaaagagcg tgcggcagaa
     4501 caggaagcgg ctcaaaaggc ccacaacgaa gccgacaatc caccgctgtt ctaacgcaat
     4561 tggggagcgg gtgtcgcggg ggttccgtgg ggggttccgt tgcaacgggt cggacaggta
     4621 aaagtcctgg tagacgctag ttttctggtt tgggccatgc ctgtctcgtt gcgtgtttcg
     4681 ttgcgcccgt tttgaatacc agccagacga gacggggttc tacgaatctt ggtcgatacc
     4741 aagccatttc cgctgaatat cggggagctc accgccagaa tcggtggttg tggtgatgta
     4801 cgtggcgaac tccgttgtag tgcctgtggt ggcatccgtg gcgcggccgc ggtaccagat
     4861 ctttaaatct agaggtgacc acaacgcgcc cgctttgatc ggggacgtct gcggccgacc
     4921 atttacgggt cttgttgtcg ttggcggtca tgggccgaac atactcaccc ggatcggagg
     4981 gccgaggaca aggtcgaacg aggggcatga cccggtgcgg ggcttcttgc actcggcata
     5041 ggcgagtgct aagaataacg ttggcactcg cgaccgggat acgactcact ataggtaccg
     5101 gtgataccag catcgtcttg atgcccttgg cagcaccctg ctaaggaggc aacaagatgc
     5161 atatgaagct caccacaatg atcaagacgg cagtagcggt cgtggccatg gcggccatcg
     5221 cgacctttgc ggcaccggtc gcgttggctg gatcc
//
