LOCUS       sequence-386116-        3403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB5r/attB1r entry clone for generation of N-terminal eGFP 
            fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3403)
  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 3403)
  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: C512-E01: attB5r-meGFP-nostop-attB1r
FEATURES             Location/Qualifiers
     source          1..3403
                     /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"
     CDS             join(313..315,316..318,319..1029)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCLSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(358..379)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(619..638)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     966..987
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1030..1154
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1226..1245)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1227..1245)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1250..1266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1250..1266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1691..2482
                     /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      2575..3163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3064..3083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3298..3325)
                     /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 caaagttgaa ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      361 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      421 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      481 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttaag ccgctacccc
      541 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      601 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      661 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      721 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
      781 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
      841 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      901 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
      961 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1021 ctgtacaaga caactttgta caaaaaagtt gaacgagaaa cgtaaaatga tataaatatc
     1081 aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat
     1141 atgcagtcac tatgaaccaa ctacttagat ggtattagtg acctgtagaa ttcgagctct
     1201 agagctgcag ggcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt
     1261 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagatcggta
     1321 cccggggatc ctctagacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt
     1381 tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg
     1441 ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt
     1501 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt
     1561 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
     1621 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
     1681 gttaaacatt atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
     1741 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
     1801 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag
     1861 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
     1921 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     1981 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     2041 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     2101 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     2161 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     2221 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     2281 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     2341 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     2401 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat
     2461 caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag
     2521 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2701 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3241 ccctgattct gtggataacc gtattaccgc tagccaggaa gagtttgtag aaacgcaaaa
     3301 aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt
     3361 cctgcccgcc accctccggg ccgttgcttc acaacgttca aat
//
