LOCUS       sequence-330541-        4188 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway attB4/attB1r entry clone for baculovirus expression of 
            His6-MBP tagged proteins using the polyhedrin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4188)
  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 4188)
  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"; journalName: "Methods Mol
            Biol."; date: "2014"; volume: "1116:193-208. doi"; pages: " 
            10.1007/978-1-62703-764-8_14"
COMMENT     Sequence Label: C417-E02: attB4-polyhedrinp>His6-MBP-nostop-at...
FEATURES             Location/Qualifiers
     source          1..4188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..45
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     37..54
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    110..205
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        271..362
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     287..304
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             369..386
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             390..1487
                     /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      1344..1353
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1461..1484
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1494..1618
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1690..1709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1691..1709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1714..1730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1714..1730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2155..2946
                     /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      3039..3627
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3528..3547
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3781..3798
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3957..3984)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4076..4162)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaaa
       61 attcgagctc ggtccgatcg ggcccgcgtt aacgctacca tggagctcca aataatgatt
      121 ttattttgac tgatagtgac ctgttcgttg caacaaattg ataagcaatg cttttttata
      181 atgccaactt tgtatagaaa agttggcgtt cgcccaggac tctagctata gttctagtgg
      241 ttggctacgt atactccgga atattaatag atcatggaga taattaaaat gataaccatc
      301 tcgcaaataa ataagtattt tactgttttc gtaacagttt tgtaataaaa aaacctataa
      361 atatgggcca ccatcaccac caccatggca aaatcgaaga aggtaaactg gtaatctgga
      421 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
      481 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
      541 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
      601 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
      661 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
      721 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
      781 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
      841 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
      901 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
      961 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
     1021 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
     1081 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
     1141 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
     1201 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     1261 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     1321 tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt gaaatcatgc
     1381 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     1441 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat tcgacaactt
     1501 tgtacaaaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     1561 ttttgcataa aaaacagact acataatact gtaaaacaca acatatgcag tcactatgaa
     1621 ccaactactt agatggtatt agtgacctgt agaattcgag ctctagagct gcagggcggc
     1681 cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg
     1741 cccgtgtctc aaaatctctg atgttacatt gcacaagatc ggtacccggg gatcctctag
     1801 accagccagg acagaaatgc ctcgacttcg ctgctaccca aggttgccgg gtgacgcaca
     1861 ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt tcgtaagctg
     1921 taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt
     1981 ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct
     2041 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     2101 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa cattatgagg
     2161 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     2221 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     2281 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     2341 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     2401 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     2461 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
     2521 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
     2581 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     2641 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     2701 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     2761 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     2821 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     2881 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     2941 aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac
     3001 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3061 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3121 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3181 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3241 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3301 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3361 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3421 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3481 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3541 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3601 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3661 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3721 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3781 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3841 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3901 gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa
     3961 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     4021 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg
     4081 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg
     4141 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcg
//
