LOCUS       sequence-357073-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Nluc(W12stop)-2xC/D box.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Verification of extracellular vesicle-mediated functional mRNA 
            delivery by RNA editing
  JOURNAL   bioRxiv 2022.01.25.477620
REFERENCE   2  (bases 1 to 6062)
  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: "bioRxiv 
            2022.01.25.477620"
COMMENT     Sequence Label: pCIneo-Nluc(W12stop)-2xC/D box
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1105..1617
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGD*RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1618..1641
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1766..1887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1777..1796)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1831..1850
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2073..2528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2160..2179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2370..2391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2645..3002
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2645..2836
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2668..2688)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2853..2988
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3053..3847
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3107..3126)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3717..3736
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3911..3959
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4000..4019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4119..4141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4179..4197)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4265..4369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4370..4438,4439..5230)
                     /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(4588..4607)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5401..5989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5890..5909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat cccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc ctcgagaatt cgccatggtc ttcacactcg aagatttcgt tggggactag
     1141 cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg
     1201 tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat
     1261 gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg
     1321 ggccagatcg aaaaaatttt taaggtggtg taccctgtgg atgatcatca ctttaaggtg
     1381 atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc
     1441 ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc
     1501 ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg
     1561 ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcggac
     1621 tacaaagacg atgacgacaa gtaaggatcc tctagagtcg acccgggcgg ggcgtgatcc
     1681 gaaaggtgac ccggatctgg ggcgtgatcc gaaaggtgac ccgccgcttc cctttagtga
     1741 gggttaatgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     1801 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1861 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1921 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     1981 atccgataag gatcgatccg ggctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2041 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     2101 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2161 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2221 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2281 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2341 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2401 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2461 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2521 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2581 cgcggatctg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     2641 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2701 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2761 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2821 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2881 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2941 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3001 aaaagcttga ttcttctgac acaacagtct cgaacttaag gctagagcca ccatgattga
     3061 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3121 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3181 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3241 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3301 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3361 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3421 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3481 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3541 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3601 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3661 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3721 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3781 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3841 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3901 cgatggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga
     3961 atcgatagcg ataaggatac gcgtatggtg cactctcagt acaatctgct ctgatgccgc
     4021 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4081 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4141 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4201 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4921 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4981 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5041 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5101 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5161 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5221 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5461 agcggtggtt tatttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgg ctcgacagat
     6061 ct
//
