LOCUS       sequence-330945-        3050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal dual 
            Strep-tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3050)
  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 3050)
  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-E67: attB5r-RU5u-Strep2x2-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(80..203)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     LTR             207..475
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             join(485..508,509..544,545..568)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     protein_bind    572..696
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(768..787)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(769..787)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(792..808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(792..808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1233..2024
                     /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      2117..2705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2606..2625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2840..2867)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2959..3045)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tggcagttcc ctactctcgc gttaacgcta ccatggagct cgaattctac agtcactaat
       61 accatctaag tagttgattc atagtgactg catatgttgt gttttacagt attatgtagt
      121 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
      181 ttcaactttt gtatacaaag tggcgagggg ctcgcatctc tccttcacgc gcccgccgcc
      241 ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg
      301 cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt
      361 gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc
      421 tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatcaccat
      481 ggggtggtca cacccgcaat tcgaaaaggg aggcggttct ggaggcggat ccggaggctc
      541 ggcttggagc catcctcagt tcgagaaggg gacaactttg tacaaaaaag ttgaacgaga
      601 aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac
      661 ataatactgt aaaacacaac atatgcagtc actatgaacc aactacttag atggtattag
      721 tgacctgtag aattcgagct ctagagctgc agggcggccg cgatatcccc tatagtgagt
      781 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
      841 gttacattgc acaagatcgg tacccgggga tcctctagac cagccaggac agaaatgcct
      901 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
      961 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     1021 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     1081 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     1141 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     1201 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     1261 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     1321 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     1381 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     1441 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     1501 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     1561 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     1621 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     1681 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     1741 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     1801 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     1861 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     1921 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     1981 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     2041 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     2101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagccagg
     2821 aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg
     2881 cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt
     2941 caaatccgct cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat
     3001 aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt atttgatgcc
//
