LOCUS       sequence-330933-        3624 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 3624)
  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 3624)
  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-E47: attB5r-Halotag-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(189..208)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(190..208)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(213..229)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(213..229)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             654..1445
                     /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      1538..2126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2027..2046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2261..2288)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2380..2466)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(2551..2674)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2678..3568
                     /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             3581..3601
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    join(3617..3624,1..117)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gtacaaaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
       61 tttgcataaa aaacagacta cataatactg taaaacacaa catatgcagt cactatgaac
      121 caactactta gatggtatta gtgacctgta gaattcgagc tctagagctg cagggcggcc
      181 gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
      241 ccgtgtctca aaatctctga tgttacattg cacaagatcg gtacccgggg atcctctaga
      301 ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac
      361 cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt
      421 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg
      481 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta
      541 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga
      601 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac attatgaggg
      661 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc
      721 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga
      781 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc
      841 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc
      901 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag
      961 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg
     1021 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg
     1081 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat
     1141 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg
     1201 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg
     1261 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg
     1321 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg
     1381 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca
     1441 aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     1501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1681 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1921 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1981 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2041 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2101 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2161 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2221 accgtattac cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat
     2281 ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
     2341 gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc
     2401 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt
     2461 tatttgatgc ctggcagttc cctactctcg cgttaacgct accatggagc tcgaattcta
     2521 cagtcactaa taccatctaa gtagttgatt catagtgact gcatatgttg tgttttacag
     2581 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
     2641 tacgtttctc gttcaacttt tgtatacaaa gtggaccatg gcagaaatcg gtactggctt
     2701 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
     2761 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
     2821 gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
     2881 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
     2941 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
     3001 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
     3061 tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac
     3121 cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
     3181 tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca
     3241 ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga
     3301 gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg
     3361 gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc
     3421 accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg
     3481 cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg
     3541 ctggctgtcg acgctcgaga tttccggcga gccaaccact gaggatctgt actttcagag
     3601 cgataacgcg atcgccacaa cttt
//
