LOCUS       sequence-290205-        7421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insect Expression of 5BoxB-6xbsMS2-HhR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7421)
  AUTHORS   Zekri L, Kuzuoglu-Ozturk D, Izaurralde E
  TITLE     GW182 proteins cause PABP dissociation from silenced miRNA targets 
            in the absence of deadenylation.
  JOURNAL   EMBO J. 2013 Apr 3;32(7):1052-65. doi: 10.1038/emboj.2013.44. Epub 
            2013 Mar 5.
  PUBMED    23463101
REFERENCE   2  (bases 1 to 7421)
  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: 
            "10.1038/emboj.2013.44"; journalName: "EMBO J"; date: "2013-04-3- 
            3"; volume: "32"; issue: "7"; pages: "1052-65"
COMMENT     Sequence Label: pAc5.1C-Fluc-5BoxB-6xbsMS2-HhR_V
FEATURES             Location/Qualifiers
     source          1..7421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..2568
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             2588..4243
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MAEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVA
                     PANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQ
                     SMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSH
                     ARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDY
                     KIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQG
                     YGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIM
                     SGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESI
                     LLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGV
                     VFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(2657..2675)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    4290..4308
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    4332..4350
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    4356..4374
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    4414..4432
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    4438..4456
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             4492..4533
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4543..4560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4583..4600)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4657..4676)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4711..4730
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_RNA        4788..4806
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4827..4845
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4866..4884
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4905..4923
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4944..4962
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        4983..5001
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        5031..5082
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     rep_origin      complement(5603..6191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5683..5702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6362..7153,7154..7222))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6985..7004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7223..7327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7395..7413
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cttcaaggaa ttaattctat attctaaaaa cacaaatgat acttctaaaa aaaatcatga
       61 atggcatcaa ctctgaatca aatctttgca gatgcaccta cttctcattt ccactgtcac
      121 atcatttttc cagatctcgc tgcctgttat gtggcccaca aaccaagaca cgttttatgg
      181 ccattaaagc tggctgatcg tcgccaaaca ccaaatacat atcaatatgt acattcgaga
      241 aagaagcgat caaagaagcg tcttcgggcg agtaggagaa tgcggaggag aaggagaacg
      301 agctgatcta gtatctctcc acaatccaat gccaactgac caactggcca tattcggagc
      361 aatttgaagc caatttccat cgcctggcga tcgctccatt cttggctata tgtttttcac
      421 cgttcccggg gccattttca aagactcgtc ggtaagataa gattgtgtca ctcgctgtct
      481 ctcttcattt gtcgaagaat gctgaggaat ttcgcgatga cgtcggcgag tattttgaag
      541 aatgagaata atttgtattt atacgaaaat cagttagtgg aattttctac aaaaacatgt
      601 tatctataga taattttgtt gcaaaatatg ttgactatga caaagattgt atgtatatac
      661 ctttaatgta ttctcatttt cttatgtatt tataatggca atgatgatac tgatgatatt
      721 ttaagatgat gccagaccac aggctgattt ctgcgtcttt tgccgaacgc agtgcatgtg
      781 cggttgttgt tttttggaat agtttcaatt ttcggactgt ccgctttgat ttcagtttct
      841 tggcttattc aaaaagcaaa gtaaagccaa aaaagcgaga tggcaatacc aaatgcggca
      901 aaacggtagt ggaaggaaag gggtgcgggg cagcggaagg aagggtgggg cggggcgtgg
      961 cggggtctgt ggctgggcgc gacgtcaccg acgttggagc cactcctttg accatgtgtg
     1021 cgtgtgtgta ttattcgtgt ctcgccactc gccggttgtt tttttctttt tatctcgctc
     1081 tctctagcgc catctcgtac gcatgctcaa cgcaccgcat gttgccgtgt cctttatgcg
     1141 tcattttggc tcgaaatagg caattattta aacaaagatt agtcaacgaa aacgctaaaa
     1201 taaataagtc tacaatatgg ttacttattg ccatgtgtgt gcagccaacg atagcaacaa
     1261 aagcaacaac acagtggctt tccctctttc actttttgtt tgcaagcgcg tgcgagcaag
     1321 acggcacgac cggcaaacgc aattacgctg acaaagagca gacgaagttt tggccgaaaa
     1381 acatcaaggc gcctgatacg aatgcatttg caataacaat tgcgatattt aatattgttt
     1441 atgaagctgt ttgacttcaa aacacacaaa aaaaaaaata aaacaaatta tttgaaagag
     1501 aattaggaat cggacagctt atcgttacgg gctaacagca caccgagacg aaatagctta
     1561 cctgacgtca cagcctctgg aagaactgcc gccaagcaga gagagagaga aaaagaggga
     1621 gagcagctta gaccgcatgt gcttgtgtgt gaggcgtctc tctcttcgtc tcctgtttgc
     1681 gcaaacgcat agactgcact gagaaaatcg attacctatt ttttatgaat gaatatttgc
     1741 actattacta ttcaaaacta ttaagatagc aatcacattc aatagccaaa tactatacca
     1801 cctgagcgat gcaacgaaat gatcaatttg agcaaaaatg ctgcatattt aggacggcat
     1861 cattatagaa atgcttcttg ctgtgtactt ttctctcgtc tggcagctgt ttcgccgtta
     1921 ttgttaaaac cggcttaagt taggtgtgtt ttctacgact agtgatgccc ctactagaag
     1981 atgtgtgttg cacaaatgtc cctgaataac caatttgaag tgcagatagc agtaaacgta
     2041 agctaatatg aatattattt aactgtaatg ttttaatatc gctggacatt actaataaac
     2101 ccactataaa cacatgtaca tatgtatgtt ttggcataca atgagtagtt ggggaaaaaa
     2161 tgtgtaaaag caccgtgacc atcacagcat aaagataacc agctgaagta tcgaatatga
     2221 gtaaccccca aattgaatca catgccgcaa ctgataggac ccatggaagt acactcttca
     2281 tggcgatata caagacacac acaagcacga acacccagtt gcggaggaaa ttctccgtaa
     2341 atgaaaaccc aatcggcgaa caattcatac ccatatatgg taaaagtttt gaacgcgact
     2401 tgagagcgga gagcattgcg gctgataagg ttttagcgct aagcgggctt tataaaacgg
     2461 gctgcgggac cagttttcat atcactaccg tttgagttct tgtgctgtgt ggatactcct
     2521 cccgacacaa agccgctcca tcagccagca gtcgtctaat ccagagaccc cggatcgggg
     2581 taccaacatg gccgaagacg ccaaaaacat aaagaaaggc ccggcaccat tctatccact
     2641 agaggatgga accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc
     2701 tggaacaatt gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt
     2761 cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag
     2821 aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt
     2881 tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag
     2941 tatgaacatt tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt
     3001 gaacgtgcaa aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga
     3061 ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa
     3121 tgaatacgat tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa
     3181 ctcctctgga tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt
     3241 cagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat
     3301 tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat
     3361 atgtggattt cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct
     3421 tcaggattac aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa
     3481 aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc
     3541 acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg
     3601 acaaggatat gggctcactg agactacatc agctattctg attacacccg agggggatga
     3661 taaaccgggc gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga
     3721 taccgggaaa acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat
     3781 tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg
     3841 gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg
     3901 cttgaagtct ttaattaaat acaaagggta tcaggtggcc cccgctgaat tggaatcgat
     3961 attgttacaa caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc
     4021 cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga
     4081 gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt
     4141 gtttgtggac gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga
     4201 gatcctcata aaggccaaga agggcggaaa gtccaaattg taagaattcg ctagcttccc
     4261 taagtccaac taccaaactg gggattcctg ggccctgaag aagggcccct cgactagtcc
     4321 aaatacaaac tgggccctga agaagggccc atatagggcc ctgaagaagg gccctatcga
     4381 ggatattatc tcgactaagt ccaactacaa actgggccct gaagaagggc ccatataggg
     4441 ccctgaagaa gggccctatc gaggatatta tctcgaggtc acccattcga aggtaagcct
     4501 atccctaacc ctctcctcgg tctcgattct acgcgtaccg gtcatcatca ccatcaccat
     4561 tgagtttaaa cccgctgatc agcctcgact gtgccttcta aggcctgagc tcgctgatca
     4621 gcctcgatcg aggatccaga catgataaga tacattgatg agtttggaca aaccacaact
     4681 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tgcggccgca
     4741 tacaaaggat ctggctgcca aactgggtct agctctagct gtagaaaaca tgaggatcac
     4801 ccatgtctgc tggacgactg tagaaaacat gaggatcacc catgtctgct gtctagctgt
     4861 agaaaacatg aggatcaccc atgtctgctg gacgactgta gaaaacatga ggatcaccca
     4921 tgtctgctgt ctagctgtag aaaacatgag gatcacccat gtctgctgga cgactgtaga
     4981 aaacatgagg atcacccatg tctgctgtct agagggccct attctacccc cctgtcaccg
     5041 gatgtgtttt ccggtctgat gagtccgtga ggacgaaaca gggaattcga tatcaagctt
     5101 atcgataccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     5161 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     5221 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     5281 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     5341 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     5401 ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca
     5461 ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca
     5521 ggcaggtaga tgacgaccat cagggacagc ttcaaggcca gcaaaaggcc aggaaccgta
     5581 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5641 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5701 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5761 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca
     5821 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5881 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5941 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6001 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6061 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6121 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6181 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6241 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6301 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6361 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6421 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6481 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6541 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6601 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6661 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6721 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6781 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6841 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6901 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6961 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7021 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7081 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7141 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7201 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7321 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     7381 cattaaccta taaaaatagg cgtatcacga ggccctttcg t
//
