LOCUS       sequence-330597-        3662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            FLAG-tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3662)
  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 3662)
  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-E13: attB5r-RU5u-His6-GST-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3662
                     /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..580
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             593..610
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             617..1264
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="PILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFE
                     LGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
                     SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM
                     DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1222..1244
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1271..1288
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    1289..1413
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1485..1504)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1486..1504)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1509..1525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1509..1525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1950..2741
                     /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      2834..3422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3323..3342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3557..3584)
                     /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 gttacagatc accatgggat cccatcacca
      601 tcaccatcac ggcgccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac
      661 tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga
      721 aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
      781 ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga
      841 caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
      901 agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac
      961 tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt
     1021 atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
     1081 cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt
     1141 ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
     1201 gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc
     1261 aaaatcggat ctggttccgc gtggatctac aactttgtac aaaaaagttg aacgagaaac
     1321 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
     1381 atactgtaaa acacaacata tgcagtcact atgaaccaac tacttagatg gtattagtga
     1441 cctgtagaat tcgagctcta gagctgcagg gcggccgcga tatcccctat agtgagtcgt
     1501 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     1561 acattgcaca agatcggtac ccggggatcc tctagaccag ccaggacaga aatgcctcga
     1621 cttcgctgct acccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa
     1681 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     1741 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     1801 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     1861 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     1921 gcagtcgccc taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga
     1981 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     2041 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     2101 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     2161 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     2221 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     2281 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     2341 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     2401 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     2461 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     2521 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     2581 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     2641 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     2701 actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc cacccatgac
     2761 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     2821 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2881 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2941 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3001 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3061 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3121 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3181 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3241 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3301 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3361 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3421 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3481 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgct agccaggaag
     3541 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct
     3601 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa
     3661 at
//
