LOCUS       sequence-213217-        6026 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 6026)
  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 6026)
  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..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..78)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     284..303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     387..406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        387..405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    406..430
                     /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             445..467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             478..519
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             520..1266
                     /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             1273..1290
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1343..1361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1357..1404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1441..1896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1528..1547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1738..1759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1989..2804)
                     /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(1989..2804)
                     /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     2712..2731
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2796..2815
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2926..3514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3415..3434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3668..3685
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3700..3842
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3768..3787)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3928..3950
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3944..4135)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4944..6026)
                     /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     5988..6007
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
       61 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
      121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      181 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      241 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      301 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      361 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
      421 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgggc
      481 aagcccatcc ccaaccccct gctgggcctg gacagcaccg gaaagtctta cccaactgtg
      541 agtgctgatt accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct
      601 gagaagagat gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac
      661 aagggcacga agaccggtgg acccttcgga accatcaagc accctgccga actggctcac
      721 agcgctaaca acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc
      781 cctattttga gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg
      841 ggtggaccta aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag
      901 ggtcgcttgc ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct
      961 atggggctta ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca
     1021 cacaaggagc gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac
     1081 tcatacttca cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac
     1141 aaggctcttt tgtctgaccc tgtattccgc cctctcgttg ataaatatgc agcggacgaa
     1201 gatgccttct ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct
     1261 gatgccctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1321 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1381 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1441 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1501 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1561 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1621 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1681 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1741 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1801 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1861 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     1921 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1981 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2041 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2101 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2161 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2221 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2281 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2341 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2401 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2461 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2521 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2581 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2641 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2701 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2761 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2821 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2881 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2941 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3001 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3061 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3121 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3181 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3241 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3301 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3361 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3421 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3481 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3541 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3601 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3661 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3721 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     3781 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3841 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3901 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3961 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4021 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4081 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4141 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4201 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4261 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4321 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4381 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4441 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4501 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4561 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4621 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4681 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4741 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4801 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4861 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4921 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4981 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5041 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5101 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5161 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5221 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5281 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5341 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5401 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5461 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5521 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5581 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5641 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5701 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5761 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5821 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5881 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5941 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6001 acatcgtata acgttactgg tttcac
//
