LOCUS       sequence-443342-        6536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual luciferase reporter for RNAi, with target site within a tunable
            ORF (version B).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6536)
  AUTHORS   Wang PY, Bartel DP
  TITLE     The guide-RNA sequence dictates the slicing kinetics and 
            conformational dynamics of the Argonaute silencing complex.
  JOURNAL   Mol Cell. 2024 Jul 10:S1097-2765(24)00533-1. doi: 
            10.1016/j.molcel.2024.06.026.
  PUBMED    39025072
REFERENCE   2  (bases 1 to 6536)
  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: "Mol Cell. 
            2024 Jul 10:S1097-2765(24)00533-1. doi: 
            10.1016/j.molcel.2024.06.026."
COMMENT     Sequence Label: pSliceRep_B_miR-196a.M3
FEATURES             Location/Qualifiers
     source          1..6536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     525..545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..720
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             733..759
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             772..798
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(1129..1702,1703..1705,1706..1714)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1295..1312)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1522..1541
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1721..1730
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1727..2239
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2249..2296
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2300..2419
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     polyA_signal    2561..2642
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2653..2670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2824..3412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2904..2923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3583..4374,4375..4443))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4206..4225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4444..4548)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4581..4662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4785..6437)
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     6353..6371
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
ORIGIN
        1 ctcgagtagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtat atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      481 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      541 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      601 gggatcctct agtcagctga cgcgtgctag cgcgggggtt tcctgcttca acagtgcttg
      661 gacggaaccc gagcttacgt agccaccatg gcgtatccat atgatgttcc agattatgct
      721 ggaggttctt cataccccta tgatgtgcct gactacgctg gcagcgccgg atacccttat
      781 gatgtgcctg attatgctac ccaaaaacat gcaattacct acggatctgc tgcgggtggc
      841 gaagaagata agaaaccggc aggtggcgaa ggtggcggtg cccatatcaa cctgaaagtg
      901 aaaggtcaag acggcaacga agtctttttc cgcatcaaac gttctaccca gctgaaaaag
      961 ctgatgaacg catactgtga ccgtcagtct gtagacatga aggcaattgc tttcctcttt
     1021 aagggtcgtc gcctacgtgc ggaaaggacc ccggatgaac tggaaatgga agatggcgac
     1081 gaaatcgacg caatgctgca ccagactgga ggcggatcct aaccgcggcc cctctccctc
     1141 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     1201 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     1261 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     1321 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     1381 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     1441 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     1501 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     1561 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     1621 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     1681 ctttgaaaaa cacgatgata atatggccac aacctacgta gccaccatgg tcttcacact
     1741 cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc aagtccttga
     1801 acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag
     1861 gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga
     1921 aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt
     1981 ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac
     2041 gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa
     2101 aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat
     2161 caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct
     2221 gtgcgaacgc attctggcgg gatcgggagc ttgcaagaac tggttcagta gcttaagcca
     2281 ctttgtgatc caccttaaca gccacggctt ccctcccgag gtggaggagc aggccgccgg
     2341 caccctgccc atgagctgcg cccaggagag cggcatggat agacaccctg ctgcttgcgc
     2401 cagcgccagg atcaacgtcc cgcatcgata agcttgtcga cgatatctcc agaggatcat
     2461 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2521 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2581 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2641 gccccgagct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2701 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2761 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2821 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2881 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2941 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3001 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3061 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3121 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3181 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3241 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3301 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3361 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3421 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3481 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3541 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3601 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3661 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3721 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3781 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3841 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3901 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3961 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4021 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4081 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4141 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     4201 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4261 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4321 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4381 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4441 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4501 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4561 aaaagtgcca cctgacgtcg gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4621 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4681 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     4741 tacaaatgtg gtatggctga ttatgatcct ctagactgca gcctttacac ggcgatcttt
     4801 ccgcccttct tggcctttat gaggatctct ctgatttttc ttgcgtcgag ttttccggta
     4861 agacctttcg gtacttcgtc cacaaacaca actcctccgc gcaacttttt cgcggttgtt
     4921 acttgactgg cgacgtaatc cacgatctct ttttccgtca tcgtctttcc gtgctccaaa
     4981 acaacaacgg cggcgggaag ttcaccggcg tcatcgtcgg gaagacctgc gacacctgcg
     5041 tcgaagatgt tggggtgttg gagcaagatg gattccaatt cagcgggagc cacctgatag
     5101 cctttgtact taatcagaga cttcaggcgg tcaacgatga agaagtgttc gtcttcgtcc
     5161 cagtaagcta tgtctccaga atgtagccat ccatccttgt caatcaaggc gttggtcgct
     5221 tccggattgt ttacataacc ggacataatc ataggacctc tcacacacag ttcgcctctt
     5281 tgattaacgc ccagcgtttt cccggtatcc agatccacaa ccttcgcttc aaaaaatgga
     5341 acaactttac cgaccgcgcc cggtttatca tccccctcgg gtgtaatcag aatagctgat
     5401 gtagtctcag tgagcccata tccttgcctg atacctggca gatggaacct cttggcaacc
     5461 gcttccccga cttccttaga gaggggagcg ccaccagaag caatttcgtg taaattagat
     5521 aaatcgtatt tgtcaatcag agtgcttttg gcgaagaagg agaatagggt tggcaccagc
     5581 agcgcacttt gaatcttgta atcctgaagg ctcctcagaa acagctcttc ttcaaatcta
     5641 tacattaaga cgactcgaaa tccacatatc aaatatccga gtgtagtaaa cattccaaaa
     5701 ccgtgatgga atggaacaac acttaaaatc gcagtatccg gaatgatttg attgccaaaa
     5761 ataggatctc tggcatgcga gaatctcacg caggcagttc tatgaggcag agcgacacct
     5821 ttaggcagac cagtagatcc agaggagttc atgatcagtg caattgtctt gtccctatcg
     5881 aaggactctg gcacaaaatc gtattcatta aaaccgggag gtagatgaga tgtgacgaac
     5941 gtgtacatcg actgaaatcc ctggtaatcc gttttagaat ccatgataat aattttttgg
     6001 atgattggga gctttttttg cacgttcaaa attttttgca accccttttt ggaaacgaac
     6061 accacggtag gctgcgaaat gcccatactg ttgagcaatt cacgttcatt ataaatgtcg
     6121 ttcgcgggcg caactgcaac tccgataaat aacgcgccca acaccggcat aaagaattga
     6181 agagagtttt cactgcatac gacgattctg tgatttgtat tcagcccata tcgtttcata
     6241 gcttctgcca accgaacgga catttcgaag tactcagcgt aagtgatgtc cacctcgata
     6301 tgtgcatctg taaaagcaat tgttccagga accagggcgt atctcttcat agccttatgc
     6361 agttgctctc cagcggttcc atcttccagc ggatagaatg gcgccgggcc tttctttatg
     6421 tttttggcgt cttccatccc gcggcatatg accggtgaat tctccaggcg atctgacggt
     6481 tcactaaacg agctctgctt atataggcct cccaccgtac acgccacctc gacata
//
