LOCUS       sequence-330937-        3845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            His6-MBP-tagged fusion proteins in bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3845)
  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 3845)
  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-E51: attB5r-RBS-His6-MBP-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(44..167)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             206..223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             227..1324
                     /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      1181..1190
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1298..1321
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1331..1455
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1527..1546)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1528..1546)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1551..1567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1551..1567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1992..2783
                     /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      2876..3464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3365..3384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3599..3626)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3718..3804)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 agctcgaatt ctacagtcac taataccatc taagtagttg attcatagtg actgcatatg
       61 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
      121 atttatatca ttttacgttt ctcgttcaac ttttgtatac aaagtggtca tttaacttta
      181 agaaggagat atacatatga gatcccatca ccatcaccat cacggcaaaa tcgaagaagg
      241 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
      301 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
      361 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
      421 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
      481 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
      541 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
      601 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
      661 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
      721 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
      781 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
      841 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
      901 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
      961 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     1021 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1081 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1141 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccaccatgg aaaacgccca
     1201 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     1261 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     1321 gactaattcg acaactttgt acaaaaaagt tgaacgagaa acgtaaaatg atataaatat
     1381 caatatatta aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca
     1441 tatgcagtca ctatgaacca actacttaga tggtattagt gacctgtaga attcgagctc
     1501 tagagctgca gggcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg
     1561 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagatcggt
     1621 acccggggat cctctagacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg
     1681 ttgccgggtg acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct
     1741 gttcggttcg taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct
     1801 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
     1861 tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
     1921 gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa
     1981 agttaaacat tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag
     2041 ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg
     2101 cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa
     2161 ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc
     2221 ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca
     2281 ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca
     2341 ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa
     2401 aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg
     2461 ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc
     2521 ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg
     2581 cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc
     2641 cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag
     2701 atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga
     2761 tcaccaaggt agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga
     2821 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2881 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2941 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3001 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3061 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3121 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3181 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3241 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3301 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3361 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3421 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3481 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3541 cccctgattc tgtggataac cgtattaccg ctagccagga agagtttgta gaaacgcaaa
     3601 aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt atggcgggcg
     3661 tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc ccggcggatt
     3721 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttc
     3781 cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgcg ttaacgctac
     3841 catgg
//
