LOCUS       sequence-231497-        7996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual luciferase assay for miRNA-targeted UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7996)
  AUTHORS   Kutsche LK, Gysi DM, Fallmann J, Lenk K, Petri R, Swiersy A, Klapper
            SD, Pircs K, Khattak S, Stadler PF, Jakobsson J, Nowick K, Busskamp 
            V
  TITLE     Combined Experimental and System-Level Analyses Reveal the Complex 
            Regulatory Network of miR-124 during Human Neurogenesis.
  JOURNAL   Cell Syst. 2018 Sep 28. pii: S2405-4712(18)30358-2. doi: 
            10.1016/j.cels.2018.08.011.
  PUBMED    30292704
REFERENCE   2  (bases 1 to 7996)
  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: "Cell Syst. 
            2018 Sep 28. pii: S2405-4712(18)30358-2. doi: 
            10.1016/j.cels.2018.08.011."
COMMENT     Sequence Label: pmiRGLO-RGS3-201
FEATURES             Location/Qualifiers
     source          1..7996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..508
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(44..67)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     441..460
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      549..558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             555..2207
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             complement(2526..2540)
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     polyA_signal    3021..3142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3032..3051)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3086..3105
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3387..3744
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3387..3578
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3410..3430)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3595..3730
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      3789..3798
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3795..4727
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             4779..5570
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4830..4849)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5440..5459
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5634..5682
                     /note="synthetic polyadenylation signal"
     promoter        5838..5942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5943..6011,6012..6803)
                     /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(6161..6180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6961..7549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7041..7060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7665..7948
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 gatctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
       61 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
      121 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
      181 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
      241 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
      301 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
      361 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
      421 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
      481 ctcctcttcc tcatctccgg gcctttcgac ctgcagccca agcttggcaa tccggtactg
      541 ttggtaaagc caccatggaa gatgccaaaa acattaagaa gggcccagcg ccattctacc
      601 cactcgaaga cgggaccgcc ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg
      661 tgcccggcac catcgccttt accgacgcac atatcgaggt ggacattacc tacgccgagt
      721 acttcgagat gagcgttcgg ctggcagaag ctatgaagcg ctatgggctg aatacaaacc
      781 atcggatcgt ggtgtgcagc gagaatagct tgcagttctt catgcccgtg ttgggtgccc
      841 tgttcatcgg tgtggctgtg gccccagcta acgacatcta caacgagcgc gagctgctga
      901 acagcatggg catcagccag cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga
      961 tcctcaacgt gcaaaagaag ctaccgatca tacaaaagat catcatcatg gatagcaaga
     1021 ccgactacca gggcttccaa agcatgtaca ccttcgtgac ttcccatttg ccacccggct
     1081 tcaacgagta cgacttcgtg cccgagagct tcgaccggga caaaaccatc gccctgatca
     1141 tgaacagtag tggcagtacc ggattgccca agggcgtagc cctaccgcac cgcaccgctt
     1201 gtgtccgatt cagtcatgcc cgcgacccca tcttcggcaa ccagatcatc cccgacaccg
     1261 ctatcctcag cgtggtgcca tttcaccacg gcttcggcat gttcaccacg ctgggctact
     1321 tgatctgcgg ctttcgggtc gtgctcatgt accgcttcga ggaggagcta ttcttgcgca
     1381 gcttgcaaga ctataagatt caatctgccc tgctggtgcc cacactattt agcttcttcg
     1441 ctaagagcac tctcatcgac aagtacgacc taagcaactt gcacgagatc gccagcggcg
     1501 gggcgccgct cagcaaggag gtaggtgagg ccgtggccaa acgcttccac ctaccaggca
     1561 tccgccaggg ctacggcctg acagaaacaa ccagcgccat tctgatcacc cccgaagggg
     1621 acgacaagcc tggcgcagta ggcaaggtgg tgcccttctt cgaggctaag gtggtggact
     1681 tggacaccgg taagacactg ggtgtgaacc agcgcggcga gctgtgcgtc cgtggcccca
     1741 tgatcatgag cggctacgtt aacaaccccg aggctacaaa cgctctcatc gacaaggacg
     1801 gctggctgca cagcggcgac atcgcctact gggacgagga cgagcacttc ttcatcgtgg
     1861 accggctgaa gagcctgatc aaatacaagg gctaccaggt agccccagcc gaactggaga
     1921 gcatcctgct gcaacacccc aacatcttcg acgccggggt cgccggcctg cccgacgacg
     1981 atgccggcga gctgcccgcc gcagtcgtcg tgctggaaca cggtaaaacc atgaccgaga
     2041 aggagatcgt ggactatgtg gccagccagg ttacaaccgc caagaagctg cgcggtggtg
     2101 ttgtgttcgt ggacgaggtg cctaaaggac tgaccggcaa gttggacgcc cgcaagatcc
     2161 gcgagattct cattaaggcc aagaagggcg gcaagatcgc cgtgtaattc tagttgttta
     2221 aacgagctcg gcaaacccaa gggcgggact ctgcttctgg caacttaacc ctttcttggg
     2281 catcccctac cacacagtaa cctcacctca actggacctg aaacagagga ccaagatggt
     2341 gggtctaggg tcatctgaag ggagtggtcc caagggtgca tgtggatgtc aacagaagga
     2401 ccacaatggt gtgagaggcc ttgtgggcat gcagagactg gcagcagaaa gtcccaagag
     2461 gagaggactc agaggcttct tccttccaag gaggaggagg aggaggacat tccacatctg
     2521 ggcttccagt ggtggctccc ggacctgcct tgggggcagc aggagaggtt gggagaggtc
     2581 accctgatga agaacaatgc catgatctgc tgctgccaga ggacatctcc gcccatccac
     2641 tcattttcca cctgtacaca ttggctcatt cattcgatgg atatttcggg tgcatctact
     2701 ttatgccagg ctctatgcta ggagctgggg atactgtgga gaaaaagtcg gatatggtct
     2761 cgctctcatc aagatttcag tctagctggg agtgagcgtt aaataagtca ataccaacac
     2821 acacacgtgc acacacgtga tggcaagctg tgatcatcta tgaaagaaaa aacacagatt
     2881 tctttaagag ggagtcgacc tgcaggcatg caagctgatc cggctgctaa caaagcccga
     2941 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcg
     3001 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     3061 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3121 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3181 gggggagatg tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     3241 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     3301 gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac catggcctga aataacctct
     3361 gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg gaatgtgtgt
     3421 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3481 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3541 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3601 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3661 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     3721 tttggaggcc taggcttttg caaaaagctt gattcttctg acacaacagt ctcgaaccaa
     3781 aggctggagc caccatggct tccaaggtgt acgaccccga gcaacgcaaa cgcatgatca
     3841 ctgggcctca gtggtgggct cgctgcaagc aaatgaacgt gctggactcc ttcatcaact
     3901 actatgattc cgagaagcac gccgagaacg ccgtgatttt tctgcatggt aacgctgcct
     3961 ccagctacct gtggaggcac gtcgtgcctc acatcgagcc cgtggctaga tgcatcatcc
     4021 ctgatctgat cggaatgggt aagtccggca agagcgggaa tggctcatat cgcctcctgg
     4081 atcactacaa gtacctcacc gcttggttcg agctgctgaa ccttccaaag aaaatcatct
     4141 ttgtgggcca cgactggggg gcttgtctgg cctttcacta ctcctacgag caccaagaca
     4201 agatcaaggc catcgtccat gctgagagtg tcgtggacgt gatcgagtcc tgggacgagt
     4261 ggcctgacat cgaggaggat atcgccctga tcaagagcga agagggcgag aaaatggtgc
     4321 ttgagaataa cttcttcgtc gagaccatgc tcccaagcaa gatcatgcgg aaactggagc
     4381 ctgaggagtt cgctgcctac ctggagccat tcaaggagaa gggcgaggtt agacggccta
     4441 ccctctcctg gcctcgcgag atccctctcg ttaagggagg caagcccgac gtcgtccaga
     4501 ttgtccgcaa ctacaacgcc taccttcggg ccagcgacga tctgcctaag atgttcatcg
     4561 agtccgaccc tgggttcttt tccaacgcta ttgtcgaggg agctaagaag ttccctaaca
     4621 ccgagttcgt gaaggtgaag ggcctccact tcagccagga ggacgctcca gatgaaatgg
     4681 gtaagtacat caagagcttc gtggagcgcg tgctgaagaa cgagcagacc ggtggtggga
     4741 gcggaggtgg cggatcaggt ggcggaggct ccggagggat tgaacaagat ggattgcacg
     4801 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4861 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4921 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4981 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     5041 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     5101 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     5161 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5221 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5281 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     5341 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     5401 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5461 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5521 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5581 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa
     5641 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gcgataagga
     5701 tcctctttgc gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg
     5761 gtcagcaccg tttctgcgga ctggctttct acgtaatggt ttcttagacg tcaggtggca
     5821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5941 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6001 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6061 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6121 ccgaagaacg ttttccaatg atgagcactt tcaaagttct gctatgtggc gcggtattat
     6181 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6241 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6301 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacta
     6361 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6421 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6481 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6541 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6601 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6661 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6721 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6781 cctcactgat taagcattgg taattcgaaa tgaccgacca agcgacgccc aaccggtatc
     6841 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6901 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6961 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7021 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7081 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7141 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7201 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7261 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7321 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7381 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     7441 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7501 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt
     7561 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7621 catgagatta tcaaaaagga tcttcaccta gatcctttta tagtccggaa atacaggaac
     7681 gcacgctgga tggcccttcg ctgggatggt gaaaccatga aaaatggcag cttcagtgga
     7741 ttaagtgggg gtaatgtggc ctgtaccctc tggttgcata ggtattcata cggttaaaat
     7801 ttatcaggcg cgattgcggc agtttttcgg gtggtttgtt gccattttta cctgtctgct
     7861 gccgtgatcg cgctgaacgc gttttagcgg tgcgtacaat taagggatta tggtaaatcc
     7921 acttactgtc tgccctcgta gccatcgaga taaaccgcag tactccggcc acgatgcgtc
     7981 cggcgtagag gatcga
//
