LOCUS       sequence-333255-        3086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB2r/attB3 entry clone for construction of C-terminal 
            triple-FLAG epitope tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3086)
  AUTHORS   Wall VE, Garvey LA, Mehalko JL, Procter LV, Esposito D
  TITLE     Combinatorial assembly of clone libraries using site-specific 
            recombination.
  JOURNAL   Methods Mol Biol. 2014;1116:193-208. doi: 
            10.1007/978-1-62703-764-8_14.
  PUBMED    24395366
REFERENCE   2  (bases 1 to 3086)
  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: "Methods Mol
            Biol."; date: "2014"; volume: "1116:193-208. doi"; pages: " 
            10.1007/978-1-62703-764-8_14"
COMMENT     Sequence Label: C234-E08: attB2r-3xFLAG-attB3
FEATURES             Location/Qualifiers
     source          1..3086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..39
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     31..48
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     32..48
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(146..269)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             281..346
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(405..424)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    complement(541..636)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1047..1838
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1931..2519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2420..2439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2673..2690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2849..2876)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2968..3054)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctagcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct taaaattcga
       61 gctcggtccg atatccttaa gctcgggccc tgcagctcta gagctcgaat tctacaggtc
      121 actaatacca tctaagtagt tggttcatag tgactgcata tgttgtgttt tacagtatta
      181 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
      241 ttctcgttca actttcttgt acaaagtggg cggaagcggc gactacaaag accacgacgg
      301 tgattataag gatcatgaca tcgattataa ggatgacgat gacaagtaat agcactggat
      361 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag
      421 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg
      481 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgtaat
      541 caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt tgttgcaacg
      601 aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggta gcgttaacgc
      661 ggccgcgata tcggtacccg gggatcctct agaccagcca ggacagaaat gcctcgactt
      721 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
      781 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
      841 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
      901 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
      961 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     1021 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
     1081 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
     1141 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
     1201 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
     1261 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
     1321 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
     1381 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
     1441 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
     1501 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
     1561 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
     1621 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
     1681 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
     1741 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
     1801 acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa
     1861 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     1921 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1981 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2041 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2101 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2161 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2221 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2281 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2341 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2401 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2461 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2521 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2581 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2641 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2701 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2761 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg
     2821 ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     2881 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     2941 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     3001 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
     3061 ggcagttccc tactctcgcg ttaacg
//
