LOCUS       sequence-75864-e        6935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of luciferase reporter mRNA in S2 cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6935)
  AUTHORS   Fukaya T, Tomari Y
  TITLE     MicroRNAs mediate gene silencing via multiple different pathways in 
            drosophila.
  JOURNAL   Mol Cell. 2012 Dec 28;48(6):825-36. doi: 
            10.1016/j.molcel.2012.09.024. Epub 2012 Nov 1.
  PUBMED    23123195
REFERENCE   2  (bases 1 to 6935)
  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"; doi: 
            "10.1016/j.molcel.2012.09.024"; journalName: "Mol Cell"; date: 
            "2012-12-28- 28"; volume: "48"; issue: "6"; pages: "825-36"
COMMENT     Sequence Label: pAWH-Rluc-let-7-Poly(A)
FEATURES             Location/Qualifiers
     source          1..6935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        140..2649
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     protein_bind    2667..2691
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2712..3647
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    3898..3946
                     /note="synthetic polyadenylation signal"
     protein_bind    complement(3986..4010)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      4018..4027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4057..4146
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     intron          4263..4328
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4458..4478
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4903..5037
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4940..4959)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4994..5013
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(5134..5202,5203..5994)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5352..5371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6165..6753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6654..6673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6907..6924
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaggcaact
       61 cgtgaaaggt aggcggatca gcggccgcag catgcaattc tatattctaa aaacacaaat
      121 gatacttcta aaaaaaaatc atgaatggca tcaactctga atcaaatctt tgcagatgca
      181 cctacttctc atttccactg tcacatcatt tttccagatc tcgctgcctg ttatgtggcc
      241 cacaaaccaa gacacgtttt atggccatta aagctggctg atcgtcgcca aacaccaaat
      301 acataatgaa tatgtacaca ttcgagaaag aagcgatcaa agaagcgtct tcgggcggag
      361 taggagaatg cggaggagaa ggagaacgag ctgatctagt atctctccac aatccaatgc
      421 caactgacca actggccata ttcggagcaa tttgaagcca atttccatcg cctggcgatc
      481 gctccattct tggctatatg tttttcaccg ttacccgggg ccattttcaa agactcgtcg
      541 gcaagataag attgtgtcac tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt
      601 tcgcgatgac gtcggcgagt attttgaaga atgagaataa tttgtattta tacgaaaatc
      661 agttagtgga attttctaca aaaacatgtt atctatagat aattttgttg caaaatatgt
      721 tgactatgac aaagattgta tgtatatacc tttaatgtat tctcattttc ttatgtattt
      781 ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa aggcttgaat
      841 ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga atattcaatt
      901 ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa gtaaagccaa
      961 aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg
     1021 cagcggaagg aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg
     1081 acgttggagc cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc
     1141 gccggttgtt tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc
     1201 tcaacgcacc gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt
     1261 atttaaacaa agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact
     1321 tattgccatg tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc
     1381 tctttcactt tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat
     1441 tacgctgaca aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg
     1501 catttgcaat aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca
     1561 cacaaaaaaa aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt
     1621 tagggtaaca acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag
     1681 agagagagag aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct
     1741 ctctcttcgt ctctgttgcg caaacgcata gactgcactg agaaaatcga ttacctattt
     1801 tttatgaatg aatatttgca ctattactat tcaaaactat taagatagca atcacattca
     1861 atagccaaat actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc
     1921 tgcatattta ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct
     1981 ggcagctgtt tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta
     2041 gtgaatgccc tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa
     2101 gtgcagatag cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat
     2161 cgctggacat tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac
     2221 aatgagtagt tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac
     2281 cagctgaagt atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga
     2341 cccatggaag tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt
     2401 tgcggaggaa attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg
     2461 gtaaaagttt tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc
     2521 taagcgggct ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc
     2581 ttgtgctgtg tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa
     2641 tccagagacc ccggatccag atatcgacaa gtttgtacaa aaaagcaggc tccgcggccg
     2701 cccccttcac catggcttcc aaggtgtacg accccgagca acgcaaacgc atgatcactg
     2761 ggcctcagtg gtgggctcgc tgcaagcaaa tgaacgtgct ggactccttc atcaactact
     2821 atgattccga gaagcacgcc gagaacgccg tgatttttct gcatggtaac gctgcctcca
     2881 gctacctgtg gaggcacgtc gtgcctcaca tcgagcccgt ggctagatgc atcatccctg
     2941 atctgatcgg aatgggtaag tccggcaaga gcgggaatgg ctcatatcgc ctcctggatc
     3001 actacaagta cctcaccgct tggttcgagc tgctgaacct tccaaagaaa atcatctttg
     3061 tgggccacga ctggggggct tgtctggcct ttcactactc ctacgagcac caagacaaga
     3121 tcaaggccat cgtccatgct gagagtgtcg tggacgtgat cgagtcctgg gacgagtggc
     3181 ctgacatcga ggaggatatc gccctgatca agagcgaaga gggcgagaaa atggtgcttg
     3241 agaataactt cttcgtcgag accatgctcc caagcaagat catgcggaaa ctggagcctg
     3301 aggagttcgc tgcctacctg gagccattca aggagaaggg cgaggttaga cggcctaccc
     3361 tctcctggcc tcgcgagatc cctctcgtta agggaggcaa gcccgacgtc gtccagattg
     3421 tccgcaacta caacgcctac cttcgggcca gcgacgatct gcctaagatg ttcatcgagt
     3481 ccgaccctgg gttcttttcc aacgctattg tcgagggagc taagaagttc cctaacaccg
     3541 agttcgtgaa ggtgaagggc ctccacttca gccaggagga cgctccagat gaaatgggta
     3601 agtacatcaa gagcttcgtg gagcgcgtgc tgaagaacga gcagtaattc taggcgatcg
     3661 ctcgagacta tacaaggatc tacctcagtc gagactatac aaggatctac ctcagtcgag
     3721 actatacaag gatctacctc agtcgagact atacaaggat ctacctcagt cgagactata
     3781 caaggatcta cctcagtcga gactatacaa ggatctacct cagtcgagac tatacaagga
     3841 tctacctcag tcgagactat acaaggatct acctcagtcg acgcggccgc tggccgcaat
     3901 aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgagga tctaaatgag
     3961 tcttcggaca agggtgggcg cgccgaccca gctttcttgt acaaagtggt gagctccgcc
     4021 accatggatc tccaccgcgg tggaggccgc atcttttacc catacgatgt tcctgactat
     4081 gcgggctatc cctatgacgt cccggactat gcaggatcct atccatatga cgttccagat
     4141 tacgctgctc atggcggaca ccggtccacg tgacgtaagc tagcaggatc tttgtgaagg
     4201 aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt taaagctcta
     4261 aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta attgtttgtg
     4321 tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat gcctttaatg
     4381 aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct actgctgact
     4441 ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag gactttcctt
     4501 cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt gcttgctttg
     4561 ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg gaaaaatatt
     4621 ctgtaacctt tataagtagg cataacagtt ataatcataa catactgttt tttcttactc
     4681 cacacaggca tagagtgtct gctattaata actatgctca aaaattgtgt acctttagct
     4741 ttttaatttg taaaggggtt aataaggaat atttgatgta tagtgccttg actagagatc
     4801 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     4861 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     4921 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4981 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcccg
     5041 tttaaactac gcgtaattca aacagggttc tggcgtcgtt ctcgtactgt tttccccagg
     5101 ccagtgcttt agcgttattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5641 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5821 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6301 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6781 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6841 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6901 gagcgcagcg agtcagtgag cgaggaagcg gaaga
//
