LOCUS       sequence-182772-        7902 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 7902)
  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 7902)
  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 143-1-Dendra2
FEATURES             Location/Qualifiers
     source          1..7902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1781..1790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3059..3751
                     /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    3862..3983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3899..3918)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3953..3972
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4107..4294,4295..4306)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4361..4381)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4361..4379)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4400..4416)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4400..4416)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4413..4435)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4424..4440
                     /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(4448..4454,4455..4472,4473..4478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4493..4514
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4631..4648)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4802..5390)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4882..4901)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5561..6352,6353..6421))
                     /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     6184..6203
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6422..6526)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6689..6710)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7106..7117,7118..7255)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gtgttttaga ttgaagtaca atatttatag gtaaaattca ccagcatatg cttaaagata
       61 gccatgctcc aggtccttgt gtccttctaa ggtctgtttg tggtttaagc tgtttgttga
      121 cacacgcttg tcatctccat ttttctgtga agttattgtc agtttattgc acactgggtt
      181 gcattaagct gcagtttttt cacaacagtt ttccacattg taacagtcag tctccaaatt
      241 attccatcag tcctcaaatt tagtcatttg aggccatgaa aacacttaaa actacatgaa
      301 ggtagtttgg cattaggttt tagacttgtt cttgtttggc atattgcaac ctgtgtaaaa
      361 atgatttgat ttttattttt tttttttttt tttcagaaag aaacggtcca aaaagagtta
      421 tgtgaacatt tatccatgtg tttaatgttt tttaaattaa aaaaaaacat ttttattaaa
      481 ccacttgtga ctaaaccttt tttagttatt ctattttgtg acgagtctga atggccaaag
      541 tactcttgaa tccaaaatct ttatagtaaa atgtttaatc ttacttgcag tgttgggtat
      601 aatttgtaac aaagttgaat tccatatctg ctgagtgaaa aagtaaagta agagattgct
      661 gtaaatattt aggttattta tttattaata caatatagaa agtaacaaaa cacaatgtaa
      721 aaaatcaata aagaaagtaa atattaaaag taatgataaa ctaatgatgt aaacaatatt
      781 agcaacaaag tttgtacaat tcagggaggg taaagaattt cacctaatag tcaaactctg
      841 tattaaataa tataaaatct ttgttcttag tctgaagagg agactgtgag agaaagctta
      901 ctggttgtta aattagaaaa tttgtaactt gcagattaaa atgatttaaa taattttata
      961 aaggccatgt ttgaaacaaa tgtaaaaaca taataaatgt taagaatttc agatattgag
     1021 attttacaat ttaaaaaaag caataaagta atagcagaga cctacactct taaagtaaat
     1081 tacccaactc tgcttactag taacttttgg aatagttact agtatccatt gaacaaatac
     1141 tagaatctaa ttaaattgtg tgagtgtcca atgaaaagat tcttgtattt aaaacagtac
     1201 cagtatctat atatgtacaa aatttagtgc tagcaaaatg caaataaata ctactgcata
     1261 taatggattg cattgtaaat cgactcgcca taatacattt atttcctatt tttactttta
     1321 acaaaaaata agttcactga gtatttcatg tcattaaatc gcattaaacc tcttgcatgc
     1381 tttatgtaca ctagtgcagt tcgctaaatg accaccggtg tcactaataa cacgatcgtt
     1441 tctgaattct gcattaaacg ccatttatac gttgctctta ttagtaaaca atttctccat
     1501 catgagctcg accagtattg catatttcat cctcttaaat ccagagttgt ttcttttaat
     1561 cactttctct tcatgataaa agcagttttt ggttttcaga agtttacaac aaacccagtc
     1621 agttattgcc tcagcaaaca ctggagcaac tgcacacttg tgaaaagctt cttgcgaaac
     1681 acaaaccttt ctctggactt ataaataaag gcattcagca gctccaccat acacctccac
     1741 tggtcttgat aaagcagata tcagcagtga tacaggtacc gccaccatgg atgaggaaat
     1801 cgctgccctg gtcgttgaca acggctccgg tatgtgcaaa gccggttttg ctggagatga
     1861 tgcccctcgt gctgttttcc cctccattgt tggacgaccc agacatcagg tacatttaat
     1921 aaaccatcct aataacatgt taatcatatt gaaccacatt aaactctcat ttgtgcattt
     1981 tacatctttt aaaaccgatt tatattcgtt tttagataat gaagtttatt aaagtgttaa
     2041 ttcacagctt tattgcttgt tttgagaatc tgcttgcata agtgattttt aaacttgatc
     2101 tgattttcgt ttgtttcgaa gttcttcagc tttgaaagtc agatcttcaa ttcacagcca
     2161 tgccatagtt ttaggataca gtataagtag acgaattggg atgcatcgta gattttatgc
     2221 tgtgtttgtt atggtttttc agtggtagag attttcagag tattattagg gttatagtca
     2281 gtgtcaggtt tatttatcgt cagcgtgatg ggcaaaaaac agctggcagt acgggctctg
     2341 gatgacagag actgaggact tcattcagac ctctggatct ttgattgaca ctgagtcgtc
     2401 tcacatgagt tcatagtctt ggttaatcaa agacggtaag atgcgaacac aagcagagtc
     2461 cggcccgatc tacagtcgtc tggcccgcgg tgcagtgctg catctctggt caactgggag
     2521 tctgagatga agcactgtag ctcgggagga caacactgtc agctcataca aacaacacac
     2581 atgcgtttac atacgtgaga ttgcttttga acagttgcct aaccatttgc tttatagggc
     2641 aaatgttggt ttttcagtca ctgcagagcc aactgctgtt actctcgccc agaaatctgc
     2701 gccaacatgg acacacccag tggagcaagc catctgattt tcatggtcag attgtaaaga
     2761 agcaattact cttggacagt ttaagcagaa aagccaccaa gtcaagtcac tggtcatatg
     2821 atgaactcga agatctaata aaagttaatt taagcgttca ttacaataaa ttcaagtgaa
     2881 tctgttttaa gctagccttt aacgttttta aattccttca aacaaattag ttcctctttg
     2941 cagttcctca tttctcggtg agctggtgct ttaatccctt tactttttct tcctctaggg
     3001 agtgatggtt ggcatgggac gaattaattc ctgcagcccg ggggtaccgg tcgccaccat
     3061 gaacaccccg ggaattaacc tgatcaagga ggacatgcgc gtgaaggtgc acatggaggg
     3121 caacgtgaac ggccacgcct tcgtgatcga gggcgagggc aagggcaagc cctacgaggg
     3181 cacccagacc gccaacctga ccgtgaagga gggcgccccc ctgcccttca gctacgacat
     3241 cctgaccacc gccgtgcact acggcaaccg ggtgttcacc aagtaccccg aggacatccc
     3301 cgactacttc aagcagagct tccccgaggg ctacagctgg gagcgcacca tgaccttcga
     3361 ggacaagggc atctgcacca tccgcagcga catcagcctg gagggcgact gcttcttcca
     3421 gaacgtgcgc ttcaagggca ccaacttccc ccccaacggc cccgtgatgc agaagaaaac
     3481 cctgaagtgg gagcccagca ccgagaagct gcacgtgcgc gacggcctgc tggtgggcaa
     3541 catcaacatg gccctgctgc tggagggcgg cggccactac ctgtgcgact tcaagaccac
     3601 ctacaaggcc aagaaggtgg tgcagctgcc cgacgcccac ttcgtggacc accgcatcga
     3661 gatcctgggc aacgacagcg actacaacaa ggtgaagctg tacgagcacg ccgtggcccg
     3721 ctacagcccc ctgcccagcc aggtgtggta atctagatca taatcagcca taccacattt
     3781 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     3841 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     3901 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3961 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     4021 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcggatc
     4081 ctgcggccgc aagggaaaat agaatgaagt gatctccaaa aaataagtac tttttgactg
     4141 taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta attaagtaaa
     4201 agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa aaataatact
     4261 taagtacagt aatcaagtaa aattactcaa gtactttaca cctctggttc ttgaccccct
     4321 accttcagca agcccagcag atccactaga tcttttgttc cctttagtga gggttaattg
     4381 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4441 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4501 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4561 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4621 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4681 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4741 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4801 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4861 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4921 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4981 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5041 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5101 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5161 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5221 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5281 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5341 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5401 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5461 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5521 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5581 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5641 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5701 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5761 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5821 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5881 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5941 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6001 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6061 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6121 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6181 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6241 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6301 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6361 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6421 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6481 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6541 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     6601 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     6661 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6721 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6781 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6841 taaagggagc ccccgattta gagcttgacg gggaaagccg gccacaatga cggctactac
     6901 atggtgccat tccttcctct ttataggaat ggagactacc tcctgtccaa caatgctctt
     6961 ggatacgagt acgcctacct gttggaccca ggtcattgca caacaccaga aatgccctct
     7021 gatctgcaaa agacgtgaat atctgttcag acacccatat ccactctgtt ccacacaggt
     7081 cagaggtttg tccaggagtt cttgacagag gtgtaaaaag tactcaaaaa ttttactcaa
     7141 gtgaaagtac aagtacttag ggaaaatttt actcaattaa aagtaaaagt atctggctag
     7201 aatcttactt gagtaaaagt aaaaaagtac tccattaaaa ttgtacttga gtattaagga
     7261 agtaaaaggc tagcaagatc tgctcgagat acatttgatt agaagagttt gacttgggtt
     7321 gattatagca aatagtggaa cacaaacaga ttttgaaata ttcttgcaaa tttatttatt
     7381 aaatccattc aaatattgtc attttttgta ctgtgtaatt tatattgatt tgctcataca
     7441 atatttacat ttcgcttaaa caatacacta ttatttatat ttaaaaatag ctatatataa
     7501 atcaaatgta tagctctttt ttacacaatt aatcaaagct aaaatatttg tcacacaaat
     7561 acagtaagcc acttcacaat gatatcttca aaatctgtgc ttgactgaat catttcattt
     7621 attgttttgt aattcacatt ataccttacg ttctccaggc acagtttgaa ttgttgataa
     7681 atgaagataa tacatctgat ttaattagtc tcctttgttt ataataactt taatctaatt
     7741 tgatagtaat aattccagca atacatccat tacaataaaa atcgcttttt tattttgcac
     7801 attagtttgc cggtatttgt ttaaagtttc accatacttt taaatcatgt gctatttgaa
     7861 aagggctgtt atgactggaa ttaaatgttt ttttttatat ca
//
