LOCUS       sequence-331914-        4044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone containing divergent AcMNPV 
            polyhedrin and p10 promoters, for use in construction of 
            polycistronic baculovirus expression vectors; allows His6-MBP fusion
            for the polyhedrin transcribed protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4044)
  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 4044)
  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: C513-E08:
            attB5r-<p10p-polyhedrinp>H6-MBP-nost...
FEATURES             Location/Qualifiers
     source          1..4044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(45..131)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(216..339)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(346..455)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        479..570
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     495..512
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             577..594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             598..1695
                     /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      1552..1561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1669..1692
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1702..1826
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1898..1917)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1899..1917)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1922..1938)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1922..1938)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2363..3154
                     /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      3247..3835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3736..3755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3970..3997)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg
       61 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt
      121 cgttttattt gatgcctggc agttccctac tctcgcgtta acgctaccat ggagctcgaa
      181 ttctacagtc actaatacca tctaagtagt tgattcatag tgactgcata tgttgtgttt
      241 tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat
      301 cattttacgt ttctcgttca acttttgtat acaaagtggt cgagtgattg taaataaaat
      361 gtaatttaca gtatagtatt ttaattaata tacaaatgat ttgataataa ttcttattta
      421 actataatat attgtgttgg gttgaattaa aggtccgtat actccggaat attaatagat
      481 catggagata attaaaatga taaccatctc gcaaataaat aagtatttta ctgttttcgt
      541 aacagttttg taataaaaaa acctataaat atgggccacc atcaccacca ccatggcaaa
      601 atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct
      661 gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat
      721 aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc
      781 tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg
      841 gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc
      901 aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg
      961 ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg
     1021 aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt
     1081 gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg
     1141 ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac
     1201 aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa
     1261 acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat
     1321 tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg
     1381 ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa
     1441 aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc
     1501 gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccaccatg
     1561 gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat
     1621 gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg
     1681 aaagacgcgc agactaattc gacaactttg tacaaaaaag ttgaacgaga aacgtaaaat
     1741 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
     1801 aaaacacaac atatgcagtc actatgaacc aactacttag atggtattag tgacctgtag
     1861 aattcgagct ctagagctgc agggcggccg cgatatcccc tatagtgagt cgtattacat
     1921 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     1981 acaagatcgg tacccgggga tcctctagac cagccaggac agaaatgcct cgacttcgct
     2041 gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg
     2101 gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg
     2161 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg
     2221 ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta
     2281 cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg
     2341 ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact
     2401 atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt
     2461 gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac
     2521 ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac
     2581 ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca
     2641 cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca
     2701 gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat
     2761 cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact
     2821 ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg
     2881 gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat
     2941 ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat
     3001 ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg
     3061 acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt
     3121 gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc
     3181 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3241 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3301 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3361 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3421 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3481 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3541 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3601 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3661 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3721 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3781 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3841 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3901 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagccagg aagagtttgt
     3961 agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt
     4021 tatggcgggc gtcctgcccg ccac
//
