LOCUS       sequence-330596-        3692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal 
            eGFP-tagged fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3692)
  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 3692)
  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-E12: attB5r-RU5u-His6-eGFP-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3692
                     /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             587..604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(605..607,608..1318)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(605..607,608..1318)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(647..668)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(908..927)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1255..1276
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1319..1443
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1515..1534)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1516..1534)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1539..1555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1539..1555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1980..2771
                     /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      2864..3452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3353..3372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3587..3614)
                     /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 accatgcacc atcaccatca
      601 ccatgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      661 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      721 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      781 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      841 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      901 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      961 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1021 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1081 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1141 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1201 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1261 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagac
     1321 aactttgtac aaaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa
     1381 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tgcagtcact
     1441 atgaaccaac tacttagatg gtattagtga cctgtagaat tcgagctcta gagctgcagg
     1501 gcggccgcga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     1561 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agatcggtac ccggggatcc
     1621 tctagaccag ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac
     1681 gcacaccgtg gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta
     1741 agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca
     1801 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac
     1861 agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt
     1921 tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta
     1981 tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg
     2041 agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg
     2101 gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa
     2161 caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg
     2221 agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt
     2281 atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta
     2341 tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac
     2401 atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg
     2461 atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg
     2521 gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca
     2581 aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc
     2641 agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct
     2701 cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag
     2761 tcggcaaata accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt
     2821 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2881 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2941 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3001 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3061 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3121 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3181 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3241 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3301 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3361 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3421 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3481 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3541 tggataaccg tattaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt
     3601 caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca
     3661 ccctccgggc cgttgcttca caacgttcaa at
//
