LOCUS       sequence-330946-        3674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            His6-Halotag-tagged fusion proteins in bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3674)
  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 3674)
  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-E54: attB5r-RBS-His6-Halotag-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3674
                     /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             203..220
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             221..1111
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1124..1144
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    1160..1284
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1356..1375)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1357..1375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1380..1396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1380..1396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1821..2612
                     /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      2705..3293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3194..3213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3428..3455)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3547..3633)
                     /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 atacatatgg gccaccatca ccatcaccat atggcagaaa tcggtactgg
      241 ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt
      301 tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt
      361 gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat
      421 cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt
      481 catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg
      541 gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc
      601 atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga
      661 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
      721 ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga
      781 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
      841 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
      901 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
      961 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat
     1021 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     1081 gcgctggctg tcgacgctcg agatttccgg cgagccaacc actgaggatc tgtactttca
     1141 gagcgataac gcgatcgcca caactttgta caaaaaagtt gaacgagaaa cgtaaaatga
     1201 tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa
     1261 aacacaacat atgcagtcac tatgaaccaa ctacttagat ggtattagtg acctgtagaa
     1321 ttcgagctct agagctgcag ggcggccgcg atatccccta tagtgagtcg tattacatgg
     1381 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     1441 aagatcggta cccggggatc ctctagacca gccaggacag aaatgcctcg acttcgctgc
     1501 tacccaaggt tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga
     1561 cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt
     1621 ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt
     1681 atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg
     1741 ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc
     1801 ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc cgaagtatcg actcaactat
     1861 cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt
     1921 acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg
     1981 tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt
     2041 cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg
     2101 acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc
     2161 gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct
     2221 tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct
     2281 ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga
     2341 actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt
     2401 ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg
     2461 agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac
     2521 aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga
     2581 aaggcgagat caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc
     2641 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2701 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2761 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2821 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     2881 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2941 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3001 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3061 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3121 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3181 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3241 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3301 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3361 ctgcgttatc ccctgattct gtggataacc gtattaccgc tagccaggaa gagtttgtag
     3421 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     3481 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     3541 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     3601 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
     3661 taacgctacc atgg
//
