LOCUS       sequence-354352-        5730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An intron is inserted into the DsRed gene with a nuclear transfer 
            signal and a 3xFlag tag. The intron can be digested with EcoRV and 
            any pre-miRNA can be inserted..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5730)
  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 5730)
  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-intron
FEATURES             Location/Qualifiers
     source          1..5730
                     /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"
     primer_bind     2591..2614
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2680..2745
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2761..2780)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2826..2881
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2880..2899)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3242..3258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3242..3258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3255..3277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3266..3282
                     /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(3290..3296,3297..3314,3315..3320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3335..3356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3415..3610
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3461..3596
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3616..3750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3653..3672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3707..3726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3818..3835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3989..4577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4069..4088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4748..5539,5540..5608))
                     /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     5371..5390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5609..5713)
                     /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 ggggggcgmg gggcggggsg gggcgaggsg
      541 gagaggtgcg 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 agggatatcg tggccgcgtc catctggtca
     2461 gaaaagacaa tctttttgtt gtcaacgttg aggtgtgaca ggcttgagag ctggccatac
     2521 acttgagtga caatgacatc cactttgcct ttctctccac agctgcccgg ctactactac
     2581 gtggacgcca agctggacat cacctcccac aacgaggact acaccatcgt ggagcagtac
     2641 gagcgcaccg agggccgcca ccacctgttc ctgggtaccg actataagga ccacgacgga
     2701 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagtaact cgagtgtaga
     2761 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc
     2821 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc
     2881 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta
     2941 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg
     3001 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac
     3061 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa
     3121 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat
     3181 agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat
     3241 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3301 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3361 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct
     3421 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3481 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3541 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3601 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3661 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3721 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     3781 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3841 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3901 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3961 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4021 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4081 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4141 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4201 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4261 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4321 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4381 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4441 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4501 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4561 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4621 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4681 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4741 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4801 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4861 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4921 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4981 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5041 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5101 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5161 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5221 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5281 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5341 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5401 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5461 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5521 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5581 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5641 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5701 taggggttcc gcgcacattt ccccgaaaag
//
