LOCUS       sequence-330543-        4179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB4/attB1r entry clone for baculovirus expression of MBP 
            tagged proteins using the polyhedrin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4179)
  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 4179)
  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: C417-E06: attB4-polyhedrinp>MBP-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..4179
                     /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        268..359
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     284..301
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             372..1469
                     /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      1326..1335
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1443..1466
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1485..1609
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1681..1700)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1682..1700)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1705..1721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1705..1721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2146..2937
                     /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      3030..3618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3519..3538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3772..3789
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3948..3975)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4067..4153)
                     /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 agttggccgc ccaggactct agctatagtt ctagtggttg
      241 gctacgtata ctccggaata ttaatagatc atggagataa ttaaaatgat aaccatctcg
      301 caaataaata agtattttac tgttttcgta acagttttgt aataaaaaaa cctataaata
      361 tgggatccgg caaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
      421 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
      481 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
      541 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
      601 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
      661 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
      721 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
      781 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
      841 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
      901 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
      961 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     1021 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     1081 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     1141 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     1201 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     1261 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     1321 ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     1381 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     1441 tcgatgaagc cctgaaagac gcgcagacta attcgggatc ctcgacaact ttgtacaaaa
     1501 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
     1561 aaaaacagac tacataatac tgtaaaacac aacatatgca gtcactatga accaactact
     1621 tagatggtat tagtgacctg tagaattcga gctctagagc tgcagggcgg ccgcgatatc
     1681 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     1741 caaaatctct gatgttacat tgcacaagat cggtacccgg ggatcctcta gaccagccag
     1801 gacagaaatg cctcgacttc gctgctaccc aaggttgccg ggtgacgcac accgtggaaa
     1861 cggatgaagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag
     1921 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
     1981 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg
     2041 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
     2101 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acattatgag ggaagcggtg
     2161 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
     2221 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
     2281 agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
     2341 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
     2401 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
     2461 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
     2521 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
     2581 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
     2641 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
     2701 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     2761 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     2821 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     2881 ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataaccc
     2941 tcgagccacc catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     3001 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3061 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3121 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3181 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3241 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3301 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3361 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3421 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3481 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3541 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3601 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3661 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3721 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3781 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3841 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3901 gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     3961 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     4021 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     4081 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     4141 ttcgttttat ttgatgcctg gcagttccct actctcgcg
//
