LOCUS       sequence-382269-        3184 bp    DNA     circular SYN 15-APR-2025
DEFINITION  3' entry vector containing streptavidin binding protein (SBP) 
            followed by a TEV cleavage site and 2 protein G copies without pA 
            for tandem affinity purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3184)
  AUTHORS   Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne 
            P
  TITLE     A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate 
            Expression Constructs with Diverse Research Applications.
  JOURNAL   PLoS One. 2016 Aug 8;11(8):e0159277. doi: 
            10.1371/journal.pone.0159277. eCollection 2016.
  PUBMED    27500400
REFERENCE   2  (bases 1 to 3184)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2016-08-8"; volume: "11(8):e0159277. doi"; pages: " 
            10.1371/journal.pone.0159277. eCollection 2016"
COMMENT     Sequence Label: p3E-SGTAP no-pA
FEATURES             Location/Qualifiers
     source          1..3184
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(88..211)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             214..327
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             334..354
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             361..381
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(757..852)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(889..908)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(890..908)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(913..929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(913..929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1031..1050)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1042..1851
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1109..1128)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1998..2586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2487..2506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2740..2757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2916..2943)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3035..3121)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3170..3184,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3184,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cctgcagctc tagagctcga attctacagg
       61 tcactaatac catctaagta gttggttcat agtgactgca tatgttgtgt tttacagtat
      121 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
      181 gtttctcgtt caactttctt gtacaaagtg gcgatggacg agaagaccac cgggtggcgg
      241 ggcggccacg ttgtggaggg tctcgctggc gagctggagc agctcagggc ccgcttggag
      301 caccatcccc aggggcaacg cgagcctatc gatgaaaatc tgtacttcca ggggatcgat
      361 gagaacctgt actttcaggg gggtagtacc ccggccgtga cgacctacaa gctggtgatc
      421 aacggcaaga ccctgaaggg cgagaccaca accaaggccg tggacgctga aaccgcagag
      481 aaggccttca agcagtacgc caacgacaat ggcgtggacg gagtgtggac ctacgacgat
      541 gccaccaaga ccttcaccgt gaccgaggtt aacacccccg cagtcaccac atacaaactg
      601 gtgatcaacg gaaaaacgct gaagggcgaa actacgacca aggctgtgga cgcggagacg
      661 gccgaaaagg cgttcaaaca gtatgccaac gataacggtg tagacggcgt ctggacatat
      721 gacgacgcga caaagacttt cactgtcact gaatagcaac tttattatac aaagttggca
      781 ttataaaaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata
      841 aaatcattat ttggagctcc atggtagcgt taacgcggcc gcgatatccc ctatagtgag
      901 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
      961 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     1021 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     1081 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     1141 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     1201 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     1261 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     1321 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     1381 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     1441 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     1501 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     1561 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     1621 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     1681 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     1741 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     1801 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     1861 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     1921 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     1981 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2041 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2101 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     2161 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2221 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2281 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2341 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2401 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2461 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2521 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2581 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2641 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2701 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2761 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2821 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg
     2881 cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     2941 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     3001 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     3061 ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt
     3121 gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc ccagtcacga
     3181 cgtt
//
