LOCUS       sequence-334513-        3408 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Template plasmid for generating C-terminal 1xFLAG 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 3408)
  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 3408)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pSU312
FEATURES             Location/Qualifiers
     source          1..3408
                     /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..1767
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(1793..1840)
                     /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             2200..2994
                     /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(2254..2273)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2864..2883
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3185..3218)
                     /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        3304..3408
                     /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 ttcgactaca aagatgacga cgataaatag taatgtaggc tggagctgct tcgaagttcc
     1801 tatactttct agagaatagg aacttcggaa taggaacttc aagatccccc acgctgccgc
     1861 aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag
     1921 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     1981 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     2041 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     2101 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     2161 tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2221 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2281 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2341 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     2401 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2461 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2521 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2581 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2641 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2701 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     2761 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2821 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2881 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2941 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3001 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3061 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3121 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccagc ttcaaaagcg
     3181 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact aaggaggata
     3241 ttcatatgcg gaattcccgg gagagctcga tgatctgcag gtggcacttt tcggggaaat
     3301 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3361 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagt
//
