LOCUS       sequence-182586-        7899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpression of microRNA and Dendra2 in Zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7899)
  AUTHORS   Hsu AY, Wang D, Liu S, Lu J, Syahirah R, Bennin DA, Huttenlocher A, 
            Umulis DM, Wan J, Deng Q
  TITLE     Phenotypical microRNA screen reveals a noncanonical role of CDK2 in 
            regulating neutrophil migration.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Aug 26. pii: 1905221116. doi: 
            10.1073/pnas.1905221116.
  PUBMED    31451657
REFERENCE   2  (bases 1 to 7899)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Aug 26. pii: 1905221116. doi: 
            10.1073/pnas.1905221116."
COMMENT     Sequence Label: Tol2-lyzC-microRNA 96--dendra2
FEATURES             Location/Qualifiers
     source          1..7899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1847..1856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3122..3814
                     /codon_start=1
                     /product="monomeric photoswitchable green-to-red 
                     fluorescent protein from Dendronephthya"
                     /label=Dendra2
                     /note="mammalian codon-optimized"
                     /translation="MNTPGINLIKEDMRVKVHMEGNVNGHAFVIEGEGKGKPYEGTQTA
                     NLTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEGYSWERTMTFEDKG
                     ICTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEKLHVRDGLLVGNIN
                     MALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDYNKVKLYEHAVARY
                     SPLPSQVW"
     polyA_signal    3925..4046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3962..3981)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4016..4035
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4170..4357,4358..4369)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4424..4444)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4424..4442)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4463..4479)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4463..4479)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4476..4498)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4487..4503
                     /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(4511..4517,4518..4535,4536..4541))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4556..4577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4694..4711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4865..5453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4945..4964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5624..6415,6416..6484))
                     /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     6247..6266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6485..6589)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6752..6773)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7169..7180,7181..7318)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 cttttaaatc atgtgctatt tgaaaagggc tgttatgact ggaattaaat gttttttttt
       61 atatcagtgt tttagattga agtacaatat ttataggtaa aattcaccag catatgctta
      121 aagatagcca tgctccaggt ccttgtgtcc ttctaaggtc tgtttgtggt ttaagctgtt
      181 tgttgacaca cgcttgtcat ctccattttt ctgtgaagtt attgtcagtt tattgcacac
      241 tgggttgcat taagctgcag ttttttcaca acagttttcc acattgtaac agtcagtctc
      301 caaattattc catcagtcct caaatttagt catttgaggc catgaaaaca cttaaaacta
      361 catgaaggta gtttggcatt aggttttaga cttgttcttg tttggcatat tgcaacctgt
      421 gtaaaaatga tttgattttt attttttttt tttttttttc agaaagaaac ggtccaaaaa
      481 gagttatgtg aacatttatc catgtgttta atgtttttta aattaaaaaa aaacattttt
      541 attaaaccac ttgtgactaa acctttttta gttattctat tttgtgacga gtctgaatgg
      601 ccaaagtact cttgaatcca aaatctttat agtaaaatgt ttaatcttac ttgcagtgtt
      661 gggtataatt tgtaacaaag ttgaattcca tatctgctga gtgaaaaagt aaagtaagag
      721 attgctgtaa atatttaggt tatttattta ttaatacaat atagaaagta acaaaacaca
      781 atgtaaaaaa tcaataaaga aagtaaatat taaaagtaat gataaactaa tgatgtaaac
      841 aatattagca acaaagtttg tacaattcag ggagggtaaa gaatttcacc taatagtcaa
      901 actctgtatt aaataatata aaatctttgt tcttagtctg aagaggagac tgtgagagaa
      961 agcttactgg ttgttaaatt agaaaatttg taacttgcag attaaaatga tttaaataat
     1021 tttataaagg ccatgtttga aacaaatgta aaaacataat aaatgttaag aatttcagat
     1081 attgagattt tacaatttaa aaaaagcaat aaagtaatag cagagaccta cactcttaaa
     1141 gtaaattacc caactctgct tactagtaac ttttggaata gttactagta tccattgaac
     1201 aaatactaga atctaattaa attgtgtgag tgtccaatga aaagattctt gtatttaaaa
     1261 cagtaccagt atctatatat gtacaaaatt tagtgctagc aaaatgcaaa taaatactac
     1321 tgcatataat ggattgcatt gtaaatcgac tcgccataat acatttattt cctattttta
     1381 cttttaacaa aaaataagtt cactgagtat ttcatgtcat taaatcgcat taaacctctt
     1441 gcatgcttta tgtacactag tgcagttcgc taaatgacca ccggtgtcac taataacacg
     1501 atcgtttctg aattctgcat taaacgccat ttatacgttg ctcttattag taaacaattt
     1561 ctccatcatg agctcgacca gtattgcata tttcatcctc ttaaatccag agttgtttct
     1621 tttaatcact ttctcttcat gataaaagca gtttttggtt ttcagaagtt tacaacaaac
     1681 ccagtcagtt attgcctcag caaacactgg agcaactgca cacttgtgaa aagcttcttg
     1741 cgaaacacaa acctttctct ggacttataa ataaaggcat tcagcagctc caccatacac
     1801 ctccactggt cttgataaag cagatatcag cagtgataca ggtaccgcca ccatggatga
     1861 ggaaatcgct gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg
     1921 agatgatgcc cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca
     1981 tttaataaac catcctaata acatgttaat catattgaac cacattaaac tctcatttgt
     2041 gcattttaca tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag
     2101 tgttaattca cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac
     2161 ttgatctgat tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca
     2221 cagccatgcc atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt
     2281 ttatgctgtg tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta
     2341 tagtcagtgt caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg
     2401 gcttgccgga gtcttaaagg gtgaagctga cagactcctg ttctgtgtat ggcactggta
     2461 gaattcactg tgaaagcaca ctatcagtga attaccaaag ggccataaac agagcagaga
     2521 aagaaccacg tgtttgattt gctctctggt gtatgggaac agattcgcaa ctcttggtcg
     2581 agctgggcgc tcttctttgc ctgttttggc actagcacat ttttgctttt ttatatatac
     2641 cttgagcaat tatgtgtagt gccaatatgg gacaagacag acatgctact taaaaaaaaa
     2701 aatcagccaa tcgcaaagga cagacagcaa gccaactgct gttactctcg cccagaaatc
     2761 tgcgccaaca tggacacacc cagtggagca agccatctga ttttcatggt cagattgtaa
     2821 agaagcaatt actcttggac agtttaagca gaaaagccac caagtcaagt cactggtcat
     2881 atgatgaact cgaagatcta ataaaagtta atttaagcgt tcattacaat aaattcaagt
     2941 gaatctgttt taagctagcc tttaacgttt ttaaattcct tcaaacaaat tagttcctct
     3001 ttgcagttcc tcatttctcg gtgagctggt gctttaatcc ctttactttt tcttcctcta
     3061 gggagtgatg gttggcatgg gacgaattaa ttcctgcagc ccgggggtac cggtcgccac
     3121 catgaacacc ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg tgcacatgga
     3181 gggcaacgtg aacggccacg ccttcgtgat cgagggcgag ggcaagggca agccctacga
     3241 gggcacccag accgccaacc tgaccgtgaa ggagggcgcc cccctgccct tcagctacga
     3301 catcctgacc accgccgtgc actacggcaa ccgggtgttc accaagtacc ccgaggacat
     3361 ccccgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca ccatgacctt
     3421 cgaggacaag ggcatctgca ccatccgcag cgacatcagc ctggagggcg actgcttctt
     3481 ccagaacgtg cgcttcaagg gcaccaactt cccccccaac ggccccgtga tgcagaagaa
     3541 aaccctgaag tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc tgctggtggg
     3601 caacatcaac atggccctgc tgctggaggg cggcggccac tacctgtgcg acttcaagac
     3661 cacctacaag gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg accaccgcat
     3721 cgagatcctg ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc acgccgtggc
     3781 ccgctacagc cccctgccca gccaggtgtg gtaatctaga tcataatcag ccataccaca
     3841 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     3901 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     3961 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4021 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
     4081 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcgg
     4141 atcctgcggc cgcaagggaa aatagaatga agtgatctcc aaaaaataag tactttttga
     4201 ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat gtaattaagt
     4261 aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc caaaaataat
     4321 acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg ttcttgaccc
     4381 cctaccttca gcaagcccag cagatccact agatcttttg ttccctttag tgagggttaa
     4441 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4501 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4561 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4621 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4681 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4741 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4801 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5701 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5761 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5821 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5881 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5941 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6001 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6061 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6121 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6181 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6241 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6301 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6361 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6601 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     6661 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6721 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6781 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6841 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6901 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggccacaa tgacggctac
     6961 tacatggtgc cattccttcc tctttatagg aatggagact acctcctgtc caacaatgct
     7021 cttggatacg agtacgccta cctgttggac ccaggtcatt gcacaacacc agaaatgccc
     7081 tctgatctgc aaaagacgtg aatatctgtt cagacaccca tatccactct gttccacaca
     7141 ggtcagaggt ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa aaattttact
     7201 caagtgaaag tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc
     7261 tagaatctta cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa
     7321 ggaagtaaaa ggctagcaag atctgctcga gatacatttg attagaagag tttgacttgg
     7381 gttgattata gcaaatagtg gaacacaaac agattttgaa atattcttgc aaatttattt
     7441 attaaatcca ttcaaatatt gtcatttttt gtactgtgta atttatattg atttgctcat
     7501 acaatattta catttcgctt aaacaataca ctattattta tatttaaaaa tagctatata
     7561 taaatcaaat gtatagctct tttttacaca attaatcaaa gctaaaatat ttgtcacaca
     7621 aatacagtaa gccacttcac aatgatatct tcaaaatctg tgcttgactg aatcatttca
     7681 tttattgttt tgtaattcac attatacctt acgttctcca ggcacagttt gaattgttga
     7741 taaatgaaga taatacatct gatttaatta gtctcctttg tttataataa ctttaatcta
     7801 atttgatagt aataattcca gcaatacatc cattacaata aaaatcgctt ttttattttg
     7861 cacattagtt tgccggtatt tgtttaaagt ttcaccata
//
