LOCUS       sequence-354350-        5848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A nontargeting miRNA based on the pre-miR- 30a structure (Qiu et 
            al., 2008) has been inserted into the EcoRV of 
            pCAG-nDsRedFL-intron..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5848)
  AUTHORS   Suzuki F, Okuno M, Tanaka T, Sanuki R
  TITLE     Overexpression of neural miRNAs miR-9/9* and miR-124 suppresses 
            differentiation to Muller glia and promotes differentiation to 
            neurons in mouse retina in vivo.
  JOURNAL   Genes Cells. 2020 Nov;25(11):741-752. doi: 10.1111/gtc.12809. Epub 
            2020 Oct 21.
  PUBMED    32979863
REFERENCE   2  (bases 1 to 5848)
  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"; doi: "10.1111/gtc.12809";
            journalName: "Genes Cells"; date: "2020-11"; volume: "25"; issue: 
            "11"; pages: "741-752"
COMMENT     Sequence Label: pCAG-nDsRedFL-miRNT
FEATURES             Location/Qualifiers
     source          1..5848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          647..1663
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1587..1609
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1671..1690
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1727..1736
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1739..1759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1943..1963)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     ncRNA           2486..2500
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     2709..2732
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2798..2863
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2879..2898)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2944..2999
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2998..3017)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3360..3376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3360..3376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3373..3395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3384..3400
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3408..3414,3415..3432,3433..3438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3453..3474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3533..3728
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3579..3714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3734..3868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3771..3790)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3825..3844
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3936..3953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4107..4695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4187..4206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4866..5657,5658..5726))
                     /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     5489..5508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5727..5831)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctgg gtcgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      421 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      481 ttttgtgcag cgatgggggc gggggggggg ggggggcgsg gggcggggsg gggsgrggsg
      541 grgrggkgsg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      601 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      661 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      721 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      781 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
      841 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
      901 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
      961 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
     1021 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     1081 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
     1141 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
     1201 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
     1261 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
     1321 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     1381 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     1441 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     1501 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     1561 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     1621 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     1681 ggttattgtg ctgtctcatc attttggcaa agaattcacc cgggaagcca ccatggctcc
     1741 aaagaagaag cgtaaggtac ggtacgcctc ctccgagaac gtcatcaccg agttcatgcg
     1801 cttcaaggtg cgcatggagg gcaccgtgaa cggccacgag ttcgagatcg agggcgaggg
     1861 cgagggccgc ccctacgagg gccacaacac cgtgaagctg aaggtgacca agggcggccc
     1921 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     1981 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     2041 ggagcgcgtg atgaacttcg aggacggcgg cgtggcgacc gtgacccagg actcctccct
     2101 gcaggacggc tgcttcatct acaaggtgaa gttcatcggc gtgaacttcc cctccgacgg
     2161 ccccgtgatg cagaagaaaa ctatgggctg ggaggcctcc accgagcgcc tgtacccccg
     2221 cgacggcgtg ctgaagggcg agacccacaa ggccctgaag ctgaaggacg gcggccacta
     2281 cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcaggtga gtactccctc
     2341 tcaaaagcgg gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg
     2401 atattcacct ggcccgcggt gatgcctttg agggatggta cctgctgttg acagtgagcg
     2461 acgatgctct aatcggttct atcaagtgaa gccacagatg ttgatagaac cttagagcat
     2521 cgctgcctac tgcctcggac ttcaagggga attcatcgtg gccgcgtcca tctggtcaga
     2581 aaagacaatc tttttgttgt caacgttgag gtgtgacagg cttgagagct ggccatacac
     2641 ttgagtgaca atgacatcca ctttgccttt ctctccacag ctgcccggct actactacgt
     2701 ggacgccaag ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga
     2761 gcgcaccgag ggccgccacc acctgttcct gggtaccgac tataaggacc acgacggaga
     2821 ctacaaggat catgatattg attacaaaga cgatgacgat aagtaactcg agtgtagatc
     2881 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     2941 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     3001 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     3061 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     3121 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     3181 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     3241 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     3301 ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc gtaatcatgg
     3361 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3421 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3481 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca
     3541 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3601 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3661 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3721 tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3781 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3841 catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg
     3901 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3961 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4021 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4081 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4141 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4201 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4261 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4321 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4381 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4441 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4501 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4561 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4621 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4681 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4741 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4801 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4861 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4921 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4981 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5041 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5101 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5161 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5221 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5281 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5341 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5401 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5461 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5521 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5581 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5641 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5701 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5761 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5821 ggggttccgc gcacatttcc ccgaaaag
//
