LOCUS       sequence-334547-        3402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid for generating C-terminal 6-His epitope tag fusions
            to bacterial genes by ""Lambda red"" recombineering..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3402)
  AUTHORS   Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L
  TITLE     Epitope tagging of chromosomal genes in Salmonella.
  JOURNAL   Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15264-9. doi: 
            10.1073/pnas.261348198. Epub 2001 Dec 11.
  PUBMED    11742086
REFERENCE   2  (bases 1 to 3402)
  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: 
            "10.1073/pnas.261348198"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2001-12-18- 18"; volume: "98"; issue: "26"; pages: "15264-9"
COMMENT     Sequence Label: pSUB7
FEATURES             Location/Qualifiers
     source          1..3402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..69,70..861)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(918..937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1078..1100
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1239..1258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      1346..1734
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             1744..1761
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(1787..1834)
                     /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             2194..2988
                     /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(2248..2267)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2858..2877
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3179..3212)
                     /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)"
     promoter        3298..3402
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      961 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     1021 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     1081 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagcaaggag
     1141 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     1201 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1261 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     1321 cggccacgat gcgtccggcg tagaggatct gaagatcagc agttcaacct gttgatagta
     1381 cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttaacgtact aagctctcat
     1441 gtttaacgaa ctaaaccctc atggctaacg tactaagctc tcatggctaa cgtactaagc
     1501 tctcatgttt cacgtactaa gctctcatgt ttgaacaata aaattaatat aaatcagcaa
     1561 cttaaatagc ctctaaggtt ttaagtttta taagaaaaaa aagaatatat aaggctttta
     1621 aagcttttaa ggtttaacgg ttgtggacaa caagccaggg atgtaacgca ctgagaagcc
     1681 cttagagcct ctcaaagcaa ttttgagtga cacaggaaca cttaacggct gacatgggaa
     1741 ttccaccacc atcatcacca ttagtaatgt aggctggagc tgcttcgaag ttcctatact
     1801 ttctagagaa taggaacttc ggaataggaa cttcaagatc ccccacgctg ccgcaagcac
     1861 tcagggcgca agggctgcta aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg
     1921 tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca
     1981 aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta
     2041 tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
     2101 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga
     2161 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     2221 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     2281 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     2341 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     2401 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     2461 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     2521 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     2581 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     2641 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     2701 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     2761 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     2821 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     2881 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     2941 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3001 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3061 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3121 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc cagcttcaaa agcgctctga
     3181 agttcctata ctctctagag aataggaact tcggaatagg aactaaggag gatattcata
     3241 tgcggaattc ccgggagagc tcgatgatct gcaggtggca cttttcgggg aaatgtgcgc
     3301 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3361 taaccctgat aaatgcttca ataatattga aaaaggaaga gt
//
