LOCUS       sequence-395647-        3561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant soluble APEX2 for SLAPSHOT labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3561)
  AUTHORS   Tuomivaara ST, Teo CF, Jan YN, Wiita AP, Jan LY
  TITLE     SLAPSHOT reveals rapid dynamics of extracellularly exposed proteome 
            in response to calcium-activated plasma membrane phospholipid 
            scrambling.
  JOURNAL   Commun Biol. 2024 Aug 29;7(1):1060. doi: 10.1038/s42003-024-06729-x.
  PUBMED    39210032
REFERENCE   2  (bases 1 to 3561)
  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: "Commun 
            Biol."; date: "2024-08-29"; volume: "7(1):1060. doi"; pages: " 
            10.1038/s42003-024-06729-x"
COMMENT     Sequence Label: pRSET-His-APEX2
FEATURES             Location/Qualifiers
     source          1..3561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     845..864
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        845..863
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             895..917
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             937..954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             964..1710
                     /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(1787..1805)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1801..1848
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1945..2400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2032..2051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2242..2263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2426..2530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2531..2599,2600..3391)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2749..2768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggatct cgatcccgcg
      841 aaattaatac gactcactat agggagacca caacggtttc cctctagaaa taattttgtt
      901 taactttaag aaggagatat acatatgcgg ggttctcatc atcatcatca tcatggtggt
      961 ggtggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
     1021 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
     1081 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
     1141 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
     1201 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
     1261 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
     1321 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat
     1381 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
     1441 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
     1501 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
     1561 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
     1621 gttgacaaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
     1681 aagctttccg agcttgggtt tgctgatgcc taactggtac catggaattc gaagcttgat
     1741 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1801 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1861 actatatccg gatctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     1921 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1981 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2041 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2101 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2161 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2221 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2281 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2341 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3241 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3541 agaaaagatc aaaggatctt c
//
