LOCUS       sequence-333254-        3535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB2r/attB3 entry clone for construction of C-terminal eGFP
            fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3535)
  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 3535)
  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: C232-E05: attB2r-meGFP-attB3
FEATURES             Location/Qualifiers
     source          1..3535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..72
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     64..81
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     65..81
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(179..302)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(305..307,308..1021)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCLSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(347..368)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(608..627)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     955..976
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1023..1118)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1529..2320
                     /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      2413..3001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2902..2921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3155..3172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3331..3358)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(join(3450..3535,1))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc ccagtcacga
       61 cgttgtaaaa cgacggccag tcttaaaatt cgagctcggt ccgatatcct taagctcggg
      121 ccctgcagct ctagagctcg aattctacag gtcactaata ccatctaagt agttggttca
      181 tagtgactgc atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
      241 atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttct tgtacaaagt
      301 gggcgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      361 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      421 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      481 cctcgtgacc accctgacct acggcgtgca gtgcttaagc cgctaccccg accacatgaa
      541 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      601 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      661 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
      721 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
      781 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
      841 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
      901 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
      961 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1021 atcaacttta ttatacaaag ttggcattat aaaaaagcat tgcttatcaa tttgttgcaa
     1081 cgaacaggtc actatcagtc aaaataaaat cattatttgg agctccatgg tagcgttaac
     1141 gcggccgcga tatcggtacc cggggatcct ctagaccagc caggacagaa atgcctcgac
     1201 ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac
     1261 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
     1321 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
     1381 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
     1441 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     1501 cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac
     1561 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     1621 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct
     1681 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     1741 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     1801 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     1861 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     1921 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     1981 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     2041 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     2101 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     2161 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     2221 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     2281 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc
     2341 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2521 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3181 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3241 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     3301 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     3361 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
     3421 ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
     3481 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tattt
//
