LOCUS       sequence-330528-        5265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB2r/attB3 entry clone for generation of polycistronic 
            ffLuc2 reporter using an internal ribosome entry sequence (IRES).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5265)
  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 5265)
  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-E03: attB2r-IRES-ffLuc2-pA-attB3
FEATURES             Location/Qualifiers
     source          1..5265
                     /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(117..240)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(266..840,841..843,844..852)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(433..450)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     660..679
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             853..2502
                     /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"
     protein_bind    complement(2760..2855)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3233..4024
                     /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      4116..4704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4605..4624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4858..4875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5034..5061)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5153..5239)
                     /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 ggtccgagga tcctacaggt cactaatacc atctaagtag ttgattcata
      121 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
      181 ttaatatatt gatatttata tcaccttacg tttctcgttc agctttcttg tacaaagtgg
      241 gcgatcaagg gatccaatct cgaggcccct ctccctcccc cccccctaac gttactggcc
      301 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
      361 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
      421 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
      481 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
      541 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
      601 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
      661 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
      721 tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa
      781 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
      841 atggccacaa ccatggaaga tgccaaaaac attaagaagg gcccagcgcc attctaccca
      901 ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca tgaagcgcta cgccctggtg
      961 cccggcacca tcgcctttac cgacgcacat atcgaggtgg acattaccta cgccgagtac
     1021 ttcgagatga gcgttcggct ggcagaagct atgaagcgct atgggctgaa tacaaaccat
     1081 cggatcgtgg tgtgcagcga gaatagcttg cagttcttca tgcccgtgtt gggtgccctg
     1141 ttcatcggtg tggctgtggc cccagctaac gacatctaca acgagcgcga gctgctgaac
     1201 agcatgggca tcagccagcc caccgtcgta ttcgtgagca agaaagggct gcaaaagatc
     1261 ctcaacgtgc aaaagaagct accgatcata caaaagatca tcatcatgga tagcaagacc
     1321 gactaccagg gcttccaaag catgtacacc ttcgtgactt cccatttgcc acccggcttc
     1381 aacgagtacg acttcgtgcc cgagagcttc gaccgggaca aaaccatcgc cctgatcatg
     1441 aacagtagtg gcagtaccgg attgcccaag ggcgtagccc taccgcaccg caccgcttgt
     1501 gtccgattca gtcatgcccg cgaccccatc ttcggcaacc agatcatccc cgacaccgct
     1561 atcctcagcg tggtgccatt tcaccacggc ttcggcatgt tcaccacgct gggctacttg
     1621 atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg aggagctatt cttgcgcagc
     1681 ttgcaagact ataagattca atctgccctg ctggtgccca cactatttag cttcttcgct
     1741 aagagcactc tcatcgacaa gtacgaccta agcaacttgc acgagatcgc cagcggcggg
     1801 gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac gcttccacct accaggcatc
     1861 cgccagggct acggcctgac agaaacaacc agcgccattc tgatcacccc cgaaggggac
     1921 gacaagcctg gcgcagtagg caaggtggtg cccttcttcg aggctaaggt ggtggacttg
     1981 gacaccggta agacactggg tgtgaaccag cgcggcgagc tgtgcgtccg tggccccatg
     2041 atcatgagcg gctacgttaa caaccccgag gctacaaacg ctctcatcga caaggacggc
     2101 tggctgcaca gcggcgacat cgcctactgg gacgaggacg agcacttctt catcgtggac
     2161 cggctgaaga gcctgatcaa atacaagggc taccaggtag ccccagccga actggagagc
     2221 atcctgctgc aacaccccaa catcttcgac gccggggtcg ccggcctgcc cgacgacgat
     2281 gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg gtaaaaccat gaccgagaag
     2341 gagatcgtgg actatgtggc cagccaggtt acaaccgcca agaagctgcg cggtggtgtt
     2401 gtgttcgtgg acgaggtgcc taaaggactg accggcaagt tggacgcccg caagatccgc
     2461 gagattctca ttaaggccaa gaagggcggc aagatcgccg tgcgggatcc accggtcgcc
     2521 acctaatgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2581 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2641 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2701 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtttaaactc
     2761 aactttatta tacaaagttg gcattatagg aaagcattgc ttatcaattt gttgcaacga
     2821 acaggtcact atcagtcaaa ataaaatcat tatttgcatg caagcttaga tcctctagac
     2881 cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc
     2941 gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta
     3001 atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg
     3061 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat
     3121 gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
     3181 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga
     3241 agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca
     3301 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa
     3361 gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg
     3421 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct
     3481 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc
     3541 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
     3601 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt
     3661 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt
     3721 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga
     3781 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc
     3841 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt
     3901 catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc
     3961 agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa
     4021 ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     4081 agcgtcagac cccgtagaaa agatcaaaga tcttcttgag atcctttttt tctgcgcgta
     4141 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4201 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4261 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4321 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4381 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4441 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4501 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4561 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4621 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4681 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4741 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4801 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4861 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     4921 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4981 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     5041 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
     5101 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc
     5161 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact
     5221 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcg
//
