LOCUS       sequence-222074-        4103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial plasmid derived from pKD4 for chromosomal insertion of 
            APEX2 gene in 5' of a gene of interest by using Datsenko.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4103)
  AUTHORS   Santin YG, Doan T, Lebrun R, Espinosa L, Journet L, Cascales E
  TITLE     In vivo TssA proximity labelling during type VI secretion biogenesis
            reveals TagA as a protein that stops and holds the sheath.
  JOURNAL   Nat Microbiol. 2018 Nov;3(11):1304-1313. doi: 
            10.1038/s41564-018-0234-3. Epub 2018 Oct 1.
  PUBMED    30275513
REFERENCE   2  (bases 1 to 4103)
  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"; doi: 
            "10.1038/s41564-018-0234-3"; journalName: "Nat Microbiol"; date: 
            "2018-11"; volume: "3"; issue: "11"; pages: "1304-1313"
COMMENT     Sequence Label: pKD4-Nter-APEX2
FEATURES             Location/Qualifiers
     source          1..4103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..34)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             62..811
                     /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="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     CDS             818..844
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     rep_origin      884..1272
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(1374..2165,2166..2234))
                     /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     1997..2016
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2235..2339)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      2429..2515
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2607..2634
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(2712..2759)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3119..3913
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3173..3192)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3783..3802
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gaagttccta tactttctag agaataggaa cttcggaata ggaactaagg aggatattca
       61 tatgggaaag tcttacccaa ctgtgagtgc tgattaccag gacgccgttg agaaggcgaa
      121 gaagaagctc agaggcttca tcgctgagaa gagatgcgct cctctaatgc tccgtttggc
      181 attccactct gctggaacct ttgacaaggg cacgaagacc ggtggaccct tcggaaccat
      241 caagcaccct gccgaactgg ctcacagcgc taacaacggt cttgacatcg ctgttaggct
      301 tttggagcca ctcaaggcgg agttccctat tttgagctac gccgatttct accagttggc
      361 tggcgttgtt gccgttgagg tcacgggtgg acctaaggtt ccattccacc ctggaagaga
      421 ggacaagcct gagccaccac cagagggtcg cttgcccgat cccactaagg gttctgacca
      481 tttgagagat gtgtttggca aagctatggg gcttactgac caagatatcg ttgctctatc
      541 tgggggtcac actattggag ctgcacacaa ggagcgttct ggatttgagg gtccctggac
      601 ctctaatcct cttattttcg acaactcata cttcacggag ttgttgagtg gtgagaagga
      661 aggtctcctt cagctacctt ctgacaaggc tcttttgtct gaccctgtat tccgccctct
      721 cgttgacaaa tatgcagcgg acgaagatgc cttctttgct gattacgctg aggctcacca
      781 aaagctttcc gagcttgggt ttgctgatgc cctgcagctg cctcccctgg agcgcctgac
      841 cctggacgca gcggccggcg gaggggacca tggctaattc ccatgtcagc cgttaagtgt
      901 tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc gttacatccc
      961 tggcttgttg tccacaaccg ttaaacctta aaagctttaa aagccttata tattcttttt
     1021 tttcttataa aacttaaaac cttagaggct atttaagttg ctgatttata ttaattttat
     1081 tgttcaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtta gccatgagag
     1141 cttagtacgt tagccatgag ggtttagttc gttaaacatg agagcttagt acgttaaaca
     1201 tgagagctta gtacgtgaaa catgagagct tagtacgtac tatcaacagg ttgaactgct
     1261 gatcttcaga tcctctacgc cggacgcatc gtggccggat cttgcggccg caaaaattaa
     1321 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1381 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1441 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1501 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1561 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1621 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1681 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1741 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa ggcggttagc
     1801 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1861 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1921 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1981 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2041 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2101 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2161 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2221 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2281 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2341 acatttcccc gaaaagtgcc acctgcatcg atggcccccg atggtagtgt ggggtctccc
     2401 catgcgagag tagggaactg ccaggcatca aataaaatga aaggctcagt cgaaagactg
     2461 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     2521 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     2581 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtg
     2641 gccagtgcca agcttgcatg cagattgcag cattacacgt cttgagcgat tgtgtaggct
     2701 ggagctgctt cgaagttcct atactttcta gagaatagga acttcggaat aggaacttca
     2761 agatccccca cgctgccgca agcactcagg gcgcaagggc tgctaaagga agcggaacac
     2821 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat
     2881 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
     2941 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
     3001 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     3061 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat
     3121 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3181 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3241 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3301 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3361 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3421 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3481 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3541 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3601 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3661 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3721 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3781 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3841 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3901 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3961 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4021 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4081 caccccagct tcaaaagcgc tct
//
