LOCUS       sequence-336760-        3464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid for generating C-terminal 3xFLAG 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 3464)
  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 3464)
  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: pSUB11
FEATURES             Location/Qualifiers
     source          1..3464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..66
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(92..139)
                     /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             499..1293
                     /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(553..572)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1163..1182
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(1484..1517)
                     /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        1603..1707
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1708..1776,1777..2568)
                     /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(1926..1945)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2625..2644)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2785..2807
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2946..2965
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      3053..3441
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 gactacaaag accatgacgg tgattataaa gatcatgata tcgactacaa agatgacgac
       61 gataaatagt aatgtaggct ggagctgctt cgaagttcct atactttcta gagaatagga
      121 acttcggaat aggaacttca agatccccca cgctgccgca agcactcagg gcgcaagggc
      181 tgctaaagga agcggaacac gtagaaagcc agtccgcaga aacggtgctg accccggatg
      241 aatgtcagct actgggctat ctggacaagg gaaatcgcaa gcgcaaagag aaagcaggta
      301 gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa
      361 ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg
      421 atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga tcaagagaca
      481 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
      541 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
      601 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
      661 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
      721 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
      781 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
      841 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
      901 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
      961 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     1021 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     1081 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     1141 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     1201 gaatgggctg accgcttcct cgtgctctac ggtatcgccg ctcccgattc gcagcgcatc
     1261 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     1321 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     1381 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     1441 tcatgctgga gttcttcgcc caccccagct tcaaaagcgc tctgaagttc ctatactttc
     1501 tagagaatag gaacttcgga ataggaacta aggaggatat tcatatgcgg aattcccggg
     1561 agagctcgat gatctgcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1621 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1681 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1741 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1801 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1861 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1921 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     1981 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2041 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2101 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2161 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2221 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2281 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2341 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2401 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2461 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2521 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2581 gtttactcat atatacttta gattgattta tggtgcactc tcagtacaat ctgctctgat
     2641 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     2701 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     2761 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     2821 caccgaaacg cgcgaggcag caaggagatg gcgcccaaca gtcccccggc cacggggcct
     2881 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     2941 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     3001 gccacgatgc gtccggcgta gaggatccgg ccacgatgcg tccggcgtag aggatctgaa
     3061 gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct
     3121 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac
     3181 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg
     3241 aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa
     3301 gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa
     3361 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac
     3421 aggaacactt aacggctgac atgggaattc cacatgtgga attc
//
