LOCUS       sequence-75863-e        7076 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 7076)
  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 7076)
  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-A114-N40-HhR
FEATURES             Location/Qualifiers
     source          1..7076
                     /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"
     misc_RNA        4059..4110
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     protein_bind    complement(4127..4151)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      4159..4168
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4198..4287
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     intron          4404..4469
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4599..4619
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5044..5178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5081..5100)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5135..5154
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             join(5275..5343,5344..6135)
                     /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(5493..5512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6306..6894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6795..6814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7048..7065
                     /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 tggccgcctg
     3901 cagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3961 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaatgc
     4021 atctggtttc ctggtctgtc tcctctcggt ttaagcttcc tgtcaccgga tgtgttttcc
     4081 ggtctgatga gtccgtgagg acgaaacagg aagggtgggc gcgccgaccc agctttcttg
     4141 tacaaagtgg tgagctccgc caccatggat ctccaccgcg gtggaggccg catcttttac
     4201 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc
     4261 tatccatatg acgttccaga ttacgctgct catggcggac accggtccac gtgacgtaag
     4321 ctagcaggat ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac
     4381 ctacagagat ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac
     4441 tactgattct aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc
     4501 agtggtggaa tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg
     4561 atgatgaggc tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag
     4621 aagaccccaa ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta
     4681 atagaactct tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca
     4741 agaaaattat ggaaaaatat tctgtaacct ttataagtag gcataacagt tataatcata
     4801 acatactgtt ttttcttact ccacacaggc atagagtgtc tgctattaat aactatgctc
     4861 aaaaattgtg tacctttagc tttttaattt gtaaaggggt taataaggaa tatttgatgt
     4921 atagtgcctt gactagagat cataatcagc cataccacat ttgtagaggt tttacttgct
     4981 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     5041 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5101 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5161 tcttatcatg tctggatccc gtttaaacta cgcgtaattc aaacagggtt ctggcgtcgt
     5221 tctcgtactg ttttccccag gccagtgctt tagcgttatt gaaaaaggaa gagtatgagt
     5281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6421 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6961 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7021 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
