LOCUS       sequence-443341-        6913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual luciferase reporter for RNAi, with target site within a tunable
            ORF (version A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6913)
  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 6913)
  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_A_miR-196a.M3
FEATURES             Location/Qualifiers
     source          1..6913
                     /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..759
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             784..1497
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             784..1494
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(811..839)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1434..1454
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    join(1504..2079,2080..2082,2083..2091)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1672..1689)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1899..1918
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2098..2107
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2104..2616
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2626..2673
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2677..2796
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     polyA_signal    2938..3019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3030..3047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3201..3789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3281..3300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3960..4751,4752..4820))
                     /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     4583..4602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4821..4925)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4958..5039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5162..6814)
                     /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     6730..6748
                     /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 ttggcagtac 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 gcggactaca aagaccacga cggtgattat
      721 aaagatcatg atatcgatta caaggacgac gacgataaga cccaaaaaca tgcaattacc
      781 tacagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
      841 gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc tacaaacgga
      901 aaactcaccc ttaaatttat ttgcactact ggaaaactac ctgttccgtg gccaacactt
      961 gtcactactc tgacctatgg tgttcaatgc ttttcccgtt atccggatca catgaaacgg
     1021 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc
     1081 aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt
     1141 aatcgtatcg agttaaaggg tattgatttt aaagaagatg gaaacattct tggacacaaa
     1201 ctcgagtaca actttaactc acacaatgta tacatcacgg cagacaaaca aaagaatgga
     1261 atcaaagcta acttcaaaat tcgccacaac gttgaagatg gttccgttca actagcagac
     1321 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     1381 ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt
     1441 cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataaccg
     1501 cggcccctct ccctcccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     1561 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     1621 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     1681 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     1741 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     1801 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     1861 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     1921 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     1981 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     2041 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac ctacgtagcc
     2101 accatggtct tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac
     2161 ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc
     2221 gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat
     2281 gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt
     2341 aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg
     2401 gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc
     2461 gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt
     2521 atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga
     2581 gtgaccggct ggcggctgtg cgaacgcatt ctggcgggat cgggagcttg caagaactgg
     2641 ttcagtagct taagccactt tgtgatccac cttaacagcc acggcttccc tcccgaggtg
     2701 gaggagcagg ccgccggcac cctgcccatg agctgcgccc aggagagcgg catggataga
     2761 caccctgctg cttgcgccag cgccaggatc aacgtcccgc atcgataagc ttgtcgacga
     2821 tatctccaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2881 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2941 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3001 aaagcatttt tttcactgcc ccgagcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3061 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4561 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4921 ccgcgcacat ttccccgaaa agtgccacct gacgtcggca gtgaaaaaaa tgctttattt
     4981 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     5041 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     5101 aaagcaagta aaacctctac aaatgtggta tggctgatta tgatcctcta gactgcagcc
     5161 tttacacggc gatctttccg cccttcttgg cctttatgag gatctctctg atttttcttg
     5221 cgtcgagttt tccggtaaga cctttcggta cttcgtccac aaacacaact cctccgcgca
     5281 actttttcgc ggttgttact tgactggcga cgtaatccac gatctctttt tccgtcatcg
     5341 tctttccgtg ctccaaaaca acaacggcgg cgggaagttc accggcgtca tcgtcgggaa
     5401 gacctgcgac acctgcgtcg aagatgttgg ggtgttggag caagatggat tccaattcag
     5461 cgggagccac ctgatagcct ttgtacttaa tcagagactt caggcggtca acgatgaaga
     5521 agtgttcgtc ttcgtcccag taagctatgt ctccagaatg tagccatcca tccttgtcaa
     5581 tcaaggcgtt ggtcgcttcc ggattgttta cataaccgga cataatcata ggacctctca
     5641 cacacagttc gcctctttga ttaacgccca gcgttttccc ggtatccaga tccacaacct
     5701 tcgcttcaaa aaatggaaca actttaccga ccgcgcccgg tttatcatcc ccctcgggtg
     5761 taatcagaat agctgatgta gtctcagtga gcccatatcc ttgcctgata cctggcagat
     5821 ggaacctctt ggcaaccgct tccccgactt ccttagagag gggagcgcca ccagaagcaa
     5881 tttcgtgtaa attagataaa tcgtatttgt caatcagagt gcttttggcg aagaaggaga
     5941 atagggttgg caccagcagc gcactttgaa tcttgtaatc ctgaaggctc ctcagaaaca
     6001 gctcttcttc aaatctatac attaagacga ctcgaaatcc acatatcaaa tatccgagtg
     6061 tagtaaacat tccaaaaccg tgatggaatg gaacaacact taaaatcgca gtatccggaa
     6121 tgatttgatt gccaaaaata ggatctctgg catgcgagaa tctcacgcag gcagttctat
     6181 gaggcagagc gacaccttta ggcagaccag tagatccaga ggagttcatg atcagtgcaa
     6241 ttgtcttgtc cctatcgaag gactctggca caaaatcgta ttcattaaaa ccgggaggta
     6301 gatgagatgt gacgaacgtg tacatcgact gaaatccctg gtaatccgtt ttagaatcca
     6361 tgataataat tttttggatg attgggagct ttttttgcac gttcaaaatt ttttgcaacc
     6421 cctttttgga aacgaacacc acggtaggct gcgaaatgcc catactgttg agcaattcac
     6481 gttcattata aatgtcgttc gcgggcgcaa ctgcaactcc gataaataac gcgcccaaca
     6541 ccggcataaa gaattgaaga gagttttcac tgcatacgac gattctgtga tttgtattca
     6601 gcccatatcg tttcatagct tctgccaacc gaacggacat ttcgaagtac tcagcgtaag
     6661 tgatgtccac ctcgatatgt gcatctgtaa aagcaattgt tccaggaacc agggcgtatc
     6721 tcttcatagc cttatgcagt tgctctccag cggttccatc ttccagcgga tagaatggcg
     6781 ccgggccttt ctttatgttt ttggcgtctt ccatcccgcg gcatatgacc ggtgaattct
     6841 ccaggcgatc tgacggttca ctaaacgagc tctgcttata taggcctccc accgtacacg
     6901 ccacctcgac ata
//
