LOCUS       sequence-330600-        4085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            FLAG-tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4085)
  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 4085)
  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: C514-E15: attB5r-RU5u-His6-MBP-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..4085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(14..100)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(185..308)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     LTR             312..580
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             587..604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             608..1705
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1562..1571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1679..1702
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1712..1836
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1908..1927)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1909..1927)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1932..1948)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1932..1948)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2373..3164
                     /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      3257..3845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3746..3765
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3980..4007)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
       61 gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact
      121 ctcgcgttaa cgctaccatg gagctcgaat tctacagtca ctaataccat ctaagtagtt
      181 gattcatagt gactgcatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
      241 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa cttttgtata
      301 caaagtggcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc
      361 atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt
      421 ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt
      481 ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct
      541 acgtctttgt ttcgttttct gttctgcgcc gttacagatc accatgcatc accatcacca
      601 tcacggcaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      661 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      721 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      781 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      841 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      901 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      961 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
     1021 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
     1081 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
     1141 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
     1201 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1261 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1321 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1381 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga
     1441 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1501 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1561 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1621 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1681 tgaagccctg aaagacgcgc agactaattc gacaactttg tacaaaaaag ttgaacgaga
     1741 aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac
     1801 ataatactgt aaaacacaac atatgcagtc actatgaacc aactacttag atggtattag
     1861 tgacctgtag aattcgagct ctagagctgc agggcggccg cgatatcccc tatagtgagt
     1921 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     1981 gttacattgc acaagatcgg tacccgggga tcctctagac cagccaggac agaaatgcct
     2041 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
     2101 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     2161 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     2221 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     2281 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     2341 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     2401 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     2461 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     2521 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     2581 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     2641 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     2701 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     2761 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     2821 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     2881 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     2941 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     3001 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     3061 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     3121 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     3181 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     3241 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3301 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3361 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3421 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3481 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3541 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3601 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3661 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3721 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3781 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3841 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3901 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagccagg
     3961 aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg
     4021 cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt
     4081 caaat
//
