LOCUS       sequence-221260-        6022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For IPTG-inducible expression of APEX2 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6022)
  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 6022)
  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: pET24b-APEX2
FEATURES             Location/Qualifiers
     source          1..6022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..133
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             134..880
                     /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             887..904
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(957..975)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      971..1018
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1055..1510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1142..1161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1352..1373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1602..2417)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1602..2417)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2325..2344
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2409..2428
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2539..3127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3028..3047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3281..3298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3313..3452
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3378..3397)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3538..3560
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3554..3745)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4554..5636)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5598..5617
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5637..5714)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5920..5939
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggcaagccc atccccaacc ccctgctggg
      121 cctggacagc accggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga
      181 gaaggcgaag aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct
      241 ccgtttggca ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt
      301 cggaaccatc aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc
      361 tgttaggctt ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta
      421 ccagttggct ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc
      481 tggaagagag gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg
      541 ttctgaccat ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt
      601 tgctctatct gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg
      661 tccctggacc tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg
      721 tgagaaggaa ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt
      781 ccgccctctc gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga
      841 ggctcaccaa aagctttccg agcttgggtt tgctgatgcc ctcgagcacc accaccacca
      901 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      961 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1021 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1081 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1141 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1201 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1261 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1321 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1381 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1441 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1501 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1561 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1621 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1681 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1741 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1801 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1861 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1921 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1981 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2041 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2101 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2161 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2221 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2281 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2341 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2401 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2461 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2521 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2581 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2641 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2701 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2761 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2821 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2881 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2941 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3001 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3061 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3121 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3181 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3241 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3301 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3361 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3421 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3481 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3541 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3601 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3661 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3721 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3781 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3841 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     3901 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3961 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4021 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4081 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4141 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4201 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4261 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4321 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4381 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4441 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4501 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4561 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4621 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4681 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4741 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4801 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     4861 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4921 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4981 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5041 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5101 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5161 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5221 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5281 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5341 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5401 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5461 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5521 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5581 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5641 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5701 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5761 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5821 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     5881 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5941 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6001 cgagatctcg atcccgcgaa at
//
