LOCUS       sequence-422677-        4920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cytosolic APEX2 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4920)
  AUTHORS   Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, 
            Ting AY
  TITLE     Directed evolution of APEX2 for electron microscopy and proximity 
            labeling.
  JOURNAL   Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
  PUBMED    25419960
REFERENCE   2  (bases 1 to 4920)
  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: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     Sequence Label: pTRC-APEX2
FEATURES             Location/Qualifiers
     source          1..4920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(93..161,162..953)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAWQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(311..330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1124..1712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1613..1632
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1866..1883
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1898..2038
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1964..1983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2124..2146
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2224..2301
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             2302..3384
                     /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     complement(2321..2340)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3616..3621,3622..3638,3639..3645)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3653..3669
                     /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)."
     primer_bind     3658..3680
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3696..3713
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3714..4460
                     /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"
     primer_bind     complement(4527..4544)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4697..4783
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4875..4902
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
       61 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      121 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      181 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      241 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      301 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgcctggcaa gagcaactcg
      361 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      421 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      481 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      541 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      601 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
      661 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
      721 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
      781 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
      841 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
      901 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
      961 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1021 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1081 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1141 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1201 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1261 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1321 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1381 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1441 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1501 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1561 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1621 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1681 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     1741 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     1801 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     1861 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     1921 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     1981 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2041 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2101 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2161 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac
     2221 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     2281 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     2341 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     2401 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     2461 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     2521 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     2581 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     2641 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2701 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2761 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2821 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2881 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2941 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     3001 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     3061 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     3121 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     3181 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     3241 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     3301 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3361 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc gcgaattgat
     3421 ctggtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     3481 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     3541 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     3601 ttctgaaatg agctgttgac aattaatcat ccggctcgta taatgtgtgg aattgtgagc
     3661 ggataacaat ttcacacagg aaacagacca tggcacacca ccaccaccac cacggaaagt
     3721 cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag aagaagctca
     3781 gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca ttccactctg
     3841 ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc aagcaccctg
     3901 ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt ttggagccac
     3961 tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct ggcgttgttg
     4021 ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag gacaagcctg
     4081 agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat ttgagagatg
     4141 tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct gggggtcaca
     4201 ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc tctaatcctc
     4261 ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc
     4321 agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc gttgacaaat
     4381 atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa aagctttccg
     4441 agcttgggtt tgctgatgcc taagatccgc tagagtcgac ctgcaggcaa gcttggctgt
     4501 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     4561 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     4621 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     4681 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     4741 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     4801 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     4861 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
//
