LOCUS       sequence-330526-        6054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB2r/attB3 entry clone for generation of polycistronic 
            ffLuc2-eGFP reporter using an internal ribosome entry sequence 
            (IRES).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6054)
  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 6054)
  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: C239-E02: attB2r-IRES-ffLuc2-eGFP-pA-attB3
FEATURES             Location/Qualifiers
     source          1..6054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..45
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     37..54
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(152..275)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(301..875,876..878,879..887)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(468..485)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     695..714
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             888..2537
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     regulatory      2553..2562
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2559..2561,2562..2564,2565..3278)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2559..2561,2562..2564,2565..3275)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2604..2625)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2865..2884)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3212..3233
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3515..3610)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4021..4812
                     /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      4905..5493
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5394..5413
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5647..5664
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5823..5850)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5942..6028)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaaa
       61 attcgagctc ggtccgatat ccttaagctc gggccctgca gctctagagc tcgaattcta
      121 caggtcacta ataccatcta agtagttggt tcatagtgac tgcatatgtt gtgttttaca
      181 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
      241 ttacgtttct cgttcaactt tcttgtacaa agtgggcgat caagggatcc aatctcgagg
      301 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
      361 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
      421 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
      481 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
      541 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
      601 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
      661 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
      721 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta
      781 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
      841 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gaagatgcca
      901 aaaacattaa gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc
      961 agctgcacaa agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg
     1021 cacatatcga ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag
     1081 aagctatgaa gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata
     1141 gcttgcagtt cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag
     1201 ctaacgacat ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg
     1261 tcgtattcgt gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga
     1321 tcatacaaaa gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt
     1381 acaccttcgt gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga
     1441 gcttcgaccg ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc
     1501 ccaagggcgt agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc
     1561 ccatcttcgg caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc
     1621 acggcttcgg catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca
     1681 tgtaccgctt cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg
     1741 ccctgctggt gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg
     1801 acctaagcaa cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg
     1861 aggccgtggc caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa
     1921 caaccagcgc cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg
     1981 tggtgccctt cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga
     2041 accagcgcgg cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc
     2101 ccgaggctac aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct
     2161 actgggacga ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca
     2221 agggctacca ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct
     2281 tcgacgccgg ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg
     2341 tcgtgctgga acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc
     2401 aggttacaac cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag
     2461 gactgaccgg caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg
     2521 gcggcaagat cgccgtgcgg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc
     2581 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2641 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2701 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2761 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2821 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2881 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2941 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     3001 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     3061 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3121 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     3181 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3241 ccgggatcac tctcggcatg gacgagctgt acaagtagta atgtgccttc tagttgccag
     3301 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3361 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3421 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3481 gctggggatg cggtgggctc tatgggttta aactcaactt tattatacaa agttggcatt
     3541 ataaaaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     3601 atcattattt ggagctccat ggtagcgtta acgcggccgc gatatcggta cccggggatc
     3661 ctctagacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga
     3721 cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt
     3781 aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
     3841 agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta
     3901 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
     3961 ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt
     4021 atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc
     4081 gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
     4141 ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
     4201 acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc
     4261 gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt
     4321 tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt
     4381 atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
     4441 catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
     4501 gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct
     4561 ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc
     4621 aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat
     4681 cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
     4741 tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta
     4801 gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg
     4861 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4921 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4981 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5041 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5101 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5161 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5221 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5281 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5341 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5401 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5461 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5521 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5581 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5641 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     5701 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5761 ggcagtgagc gcaacgcaat taatacgcgt accgctagcc aggaagagtt tgtagaaacg
     5821 caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg
     5881 ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg
     5941 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
     6001 cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgcg
//
