LOCUS       sequence-333257-        3694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB2r/attB3 entry clone for construction of C-terminal 
            Halotag fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3694)
  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 3694)
  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: C234-E26: attB2r-Halotag-attB3
FEATURES             Location/Qualifiers
     source          1..3694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(52..138)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     187..209
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     201..218
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     202..218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(281..404)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             410..1297
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             1310..1330
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    complement(1353..1448)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1826..2617
                     /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      2709..3297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3198..3217
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3451..3468
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3627..3654)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
       61 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
      121 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
      181 tgttttccca gtcacgacgt tgtaaaacga cggccagtct taaaattcga gctcggtccg
      241 aggatcctac aggtcactaa taccatctaa gtagttgatt catagtgact ggatatgttg
      301 tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt
      361 tatatcacct tacgtttctc gttcagcttt cttgtacaaa gtgggcggcg cagaaatcgg
      421 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
      481 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
      541 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
      601 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
      661 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
      721 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
      781 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
      841 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
      901 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
      961 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1021 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1081 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1141 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1201 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1261 gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta
     1321 ctttcagagc gataacgcga tcgcctaata atcaacttta ttatacaaag ttggcattat
     1381 aggaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     1441 cattatttgc atgcaagctt agatcctcta gaccagccag gacagaaatg cctcgacttc
     1501 gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca
     1561 gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa
     1621 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
     1681 ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg
     1741 ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag
     1801 tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca
     1861 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
     1921 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
     1981 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
     2041 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
     2101 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
     2161 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
     2221 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
     2281 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
     2341 atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg
     2401 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
     2461 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
     2521 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
     2581 cgtgaaaggc gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa
     2641 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2701 agatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2761 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2821 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2881 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2941 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3001 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3061 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3121 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3181 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3241 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3301 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3361 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3421 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3481 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     3541 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct
     3601 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
     3661 tttgatgcct ggcagtttat ggcgggcgtc ctgc
//
