LOCUS       sequence-454990-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-luciferase reporter containing 4 copies of the 8mer site of 
            human miR-302a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7449)
  AUTHORS   Cheng ECK, Lam JKC, Kwon SC
  TITLE     Cytosolic CRISPR RNAs for efficient application of RNA-targeting 
            CRISPR-Cas systems.
  JOURNAL   EMBO Rep. 2025 Feb 26. doi: 10.1038/s44319-025-00399-4.
  PUBMED    40011676
REFERENCE   2  (bases 1 to 7449)
  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: "EMBO Rep. 
            2025 Feb 26. doi: 10.1038/s44319-025-00399-4."
COMMENT     Sequence Label: pmirGLO-mir302-8mer4x (pEZ185)
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        154..511
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        154..345
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(177..197)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      362..497
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      556..565
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             562..1494
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1546..2337
                     /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(1597..1616)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2207..2226
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2401..2449
                     /note="synthetic polyadenylation signal"
     promoter        2605..2709
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2710..2778,2779..3570)
                     /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(2928..2947)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3728..4316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3808..3827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4432..4715
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     promoter        4772..5271
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4807..4830)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5204..5223
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      5312..5321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5318..6970
                     /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"
     polyA_signal    7237..7358
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7248..7267)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7302..7321
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 aaatcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
       61 cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat
      121 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
      181 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      241 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
      301 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
      361 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
      421 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
      481 aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca caacagtctc
      541 gaaccaaagg ctggagccac catggcttcc aaggtgtacg accccgagca acgcaaacgc
      601 atgatcactg ggcctcagtg gtgggctcgc tgcaagcaaa tgaacgtgct ggactccttc
      661 atcaactact atgattccga gaagcacgcc gagaacgccg tgatttttct gcatggtaac
      721 gctgcctcca gctacctgtg gaggcacgtc gtgcctcaca tcgagcccgt ggctagatgc
      781 atcatccctg atctgatcgg aatgggtaag tccggcaaga gcgggaatgg ctcatatcgc
      841 ctcctggatc actacaagta cctcaccgct tggttcgagc tgctgaacct tccaaagaaa
      901 atcatctttg tgggccacga ctggggggct tgtctggcct ttcactactc ctacgagcac
      961 caagacaaga tcaaggccat cgtccatgct gagagtgtcg tggacgtgat cgagtcctgg
     1021 gacgagtggc ctgacatcga ggaggatatc gccctgatca agagcgaaga gggcgagaaa
     1081 atggtgcttg agaataactt cttcgtcgag accatgctcc caagcaagat catgcggaaa
     1141 ctggagcctg aggagttcgc tgcctacctg gagccattca aggagaaggg cgaggttaga
     1201 cggcctaccc tctcctggcc tcgcgagatc cctctcgtta agggaggcaa gcccgacgtc
     1261 gtccagattg tccgcaacta caacgcctac cttcgggcca gcgacgatct gcctaagatg
     1321 ttcatcgagt ccgaccctgg gttcttttcc aacgctattg tcgagggagc taagaagttc
     1381 cctaacaccg agttcgtgaa ggtgaagggc ctccacttca gccaggagga cgctccagat
     1441 gaaatgggta agtacatcaa gagcttcgtg gagcgcgtgc tgaagaacga gcagaccggt
     1501 ggtgggagcg gaggtggcgg atcaggtggc ggaggctccg gagggattga acaagatgga
     1561 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     1621 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     1681 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     1741 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     1801 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     1861 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     1921 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     1981 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     2041 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     2101 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     2161 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     2221 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     2281 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     2341 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgatggccgc
     2401 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagcg
     2461 ataaggatcc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc
     2521 atccggggtc agcaccgttt ctgcggactg gctttctacg taatggtttc ttagacgtca
     2581 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2641 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2701 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2761 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2821 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2881 tttcgccccg aagaacgttt tccaatgatg agcactttca aagttctgct atgtggcgcg
     2941 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3001 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3061 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3121 acaactatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3181 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3241 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3301 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3361 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3421 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3481 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3541 ataggtgcct cactgattaa gcattggtaa ttcgaaatga ccgaccaagc gacgcccaac
     3601 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3661 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3721 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3781 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3841 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3901 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3961 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4021 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4081 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4141 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4201 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tttcaagaag
     4321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4381 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttatag tccggaaata
     4441 caggaacgca cgctggatgg cccttcgctg ggatggtgaa accatgaaaa atggcagctt
     4501 cagtggatta agtgggggta atgtggcctg taccctctgg ttgcataggt attcatacgg
     4561 ttaaaattta tcaggcgcga ttgcggcagt ttttcgggtg gtttgttgcc atttttacct
     4621 gtctgctgcc gtgatcgcgc tgaacgcgtt ttagcggtgc gtacaattaa gggattatgg
     4681 taaatccact tactgtctgc cctcgtagcc atcgagataa accgcagtac tccggccacg
     4741 atgcgtccgg cgtagaggat cgagatctac cgggtagggg aggcgctttt cccaaggcag
     4801 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     4861 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     4921 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     4981 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     5041 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     5101 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     5161 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     5221 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc
     5281 ccaagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca aaaacattaa
     5341 gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc agctgcacaa
     5401 agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg cacatatcga
     5461 ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag aagctatgaa
     5521 gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata gcttgcagtt
     5581 cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag ctaacgacat
     5641 ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg tcgtattcgt
     5701 gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa
     5761 gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt acaccttcgt
     5821 gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg
     5881 ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc ccaagggcgt
     5941 agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg
     6001 caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc acggcttcgg
     6061 catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca tgtaccgctt
     6121 cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg ccctgctggt
     6181 gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg acctaagcaa
     6241 cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc
     6301 caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa caaccagcgc
     6361 cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg tggtgccctt
     6421 cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga accagcgcgg
     6481 cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc ccgaggctac
     6541 aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct actgggacga
     6601 ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca agggctacca
     6661 ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct tcgacgccgg
     6721 ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga
     6781 acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc aggttacaac
     6841 cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg
     6901 caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg gcggcaagat
     6961 cgccgtgtaa ttctagttgt ttaaacgagc tcgctagctt tatacgcaag ccgagcactt
     7021 attgccttta tacgcaagcc gagcacttat tttgtcgacc tgcaggcatg caagctgatc
     7081 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     7141 tagcataacc ccttggggcg gccgctacgc aagccgagca cttattgcct ttatacgcaa
     7201 gccgagcact tatgcggccg cttcgagcag acatgataag atacattgat gagtttggac
     7261 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     7321 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     7381 ttatgtttca ggttcagggg gagatgtggg aggttttttt aagcaagtaa aacctctaca
     7441 aatgtggta
//
