LOCUS       sequence-330598-        3919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            Halotag-tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3919)
  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 3919)
  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-E14: attB5r-RU5u-His6-Halotag-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(14..100)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(185..308)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     LTR             312..582
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             589..606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             607..1497
                     /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             1510..1530
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    1546..1670
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1742..1761)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1743..1761)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1766..1782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1766..1782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2207..2998
                     /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      3091..3679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3580..3599
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3814..3841)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
       61 gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact
      121 ctcgcgttaa cgctaccatg gagctcgaat tctacagtca ctaataccat ctaagtagtt
      181 gattcatagt gactgcatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
      241 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa cttttgtata
      301 caaagtggcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc
      361 atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt
      421 ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt
      481 ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct
      541 acgtctttgt ttcgttttct gttctgcgcc gggttacaga tcaccatgca ccatcaccat
      601 caccatatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
      661 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
      721 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc
      781 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
      841 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
      901 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
      961 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
     1021 gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc
     1081 cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc
     1141 cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac
     1201 cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc
     1261 gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg
     1321 ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc
     1381 ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac
     1441 ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag
     1501 ccaaccactg aggatctgta ctttcagagc gataacgcga tcgccacaac tttgtacaaa
     1561 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     1621 aaaaaacaga ctacataata ctgtaaaaca caacatatgc agtcactatg aaccaactac
     1681 ttagatggta ttagtgacct gtagaattcg agctctagag ctgcagggcg gccgcgatat
     1741 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     1801 tcaaaatctc tgatgttaca ttgcacaaga tcggtacccg gggatcctct agaccagcca
     1861 ggacagaaat gcctcgactt cgctgctacc caaggttgcc gggtgacgca caccgtggaa
     1921 acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa
     1981 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     2041 cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg
     2101 ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa
     2161 cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacattatga gggaagcggt
     2221 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
     2281 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
     2341 cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
     2401 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
     2461 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
     2521 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
     2581 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
     2641 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
     2701 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
     2761 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
     2821 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
     2881 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
     2941 gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc
     3001 ctcgagccac ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     3061 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3121 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3181 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3241 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3301 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3361 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3421 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3481 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3541 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3601 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3661 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3721 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3781 taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc
     3841 tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
     3901 tgcttcacaa cgttcaaat
//
