LOCUS       sequence-182600-        7872 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 7872)
  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 7872)
  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 141-dendra2
FEATURES             Location/Qualifiers
     source          1..7872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1721..1730
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2969..3661
                     /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    3772..3893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3809..3828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3863..3882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4017..4204,4205..4216)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4271..4291)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4271..4289)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4310..4326)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4310..4326)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4323..4345)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4334..4350
                     /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(4358..4364,4365..4382,4383..4388))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4403..4424
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4541..4558)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4712..5300)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4792..4811)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5471..6262,6263..6331))
                     /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     6094..6113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6332..6436)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6599..6620)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7016..7027,7028..7165)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gccatgctcc aggtccttgt gtccttctaa ggtctgtttg tggtttaagc tgtttgttga
       61 cacacgcttg tcatctccat ttttctgtga agttattgtc agtttattgc acactgggtt
      121 gcattaagct gcagtttttt cacaacagtt ttccacattg taacagtcag tctccaaatt
      181 attccatcag tcctcaaatt tagtcatttg aggccatgaa aacacttaaa actacatgaa
      241 ggtagtttgg cattaggttt tagacttgtt cttgtttggc atattgcaac ctgtgtaaaa
      301 atgatttgat ttttattttt tttttttttt tttcagaaag aaacggtcca aaaagagtta
      361 tgtgaacatt tatccatgtg tttaatgttt tttaaattaa aaaaaaacat ttttattaaa
      421 ccacttgtga ctaaaccttt tttagttatt ctattttgtg acgagtctga atggccaaag
      481 tactcttgaa tccaaaatct ttatagtaaa atgtttaatc ttacttgcag tgttgggtat
      541 aatttgtaac aaagttgaat tccatatctg ctgagtgaaa aagtaaagta agagattgct
      601 gtaaatattt aggttattta tttattaata caatatagaa agtaacaaaa cacaatgtaa
      661 aaaatcaata aagaaagtaa atattaaaag taatgataaa ctaatgatgt aaacaatatt
      721 agcaacaaag tttgtacaat tcagggaggg taaagaattt cacctaatag tcaaactctg
      781 tattaaataa tataaaatct ttgttcttag tctgaagagg agactgtgag agaaagctta
      841 ctggttgtta aattagaaaa tttgtaactt gcagattaaa atgatttaaa taattttata
      901 aaggccatgt ttgaaacaaa tgtaaaaaca taataaatgt taagaatttc agatattgag
      961 attttacaat ttaaaaaaag caataaagta atagcagaga cctacactct taaagtaaat
     1021 tacccaactc tgcttactag taacttttgg aatagttact agtatccatt gaacaaatac
     1081 tagaatctaa ttaaattgtg tgagtgtcca atgaaaagat tcttgtattt aaaacagtac
     1141 cagtatctat atatgtacaa aatttagtgc tagcaaaatg caaataaata ctactgcata
     1201 taatggattg cattgtaaat cgactcgcca taatacattt atttcctatt tttactttta
     1261 acaaaaaata agttcactga gtatttcatg tcattaaatc gcattaaacc tcttgcatgc
     1321 tttatgtaca ctagtgcagt tcgctaaatg accaccggtg tcactaataa cacgatcgtt
     1381 tctgaattct gcattaaacg ccatttatac gttgctctta ttagtaaaca atttctccat
     1441 catgagctcg accagtattg catatttcat cctcttaaat ccagagttgt ttcttttaat
     1501 cactttctct tcatgataaa agcagttttt ggttttcaga agtttacaac aaacccagtc
     1561 agttattgcc tcagcaaaca ctggagcaac tgcacacttg tgaaaagctt cttgcgaaac
     1621 acaaaccttt ctctggactt ataaataaag gcattcagca gctccaccat acacctccac
     1681 tggtcttgat aaagcagata tcagcagtga tacaggtacc gccaccatgg atgaggaaat
     1741 cgctgccctg gtcgttgaca acggctccgg tatgtgcaaa gccggttttg ctggagatga
     1801 tgcccctcgt gctgttttcc cctccattgt tggacgaccc agacatcagg tacatttaat
     1861 aaaccatcct aataacatgt taatcatatt gaaccacatt aaactctcat ttgtgcattt
     1921 tacatctttt aaaaccgatt tatattcgtt tttagataat gaagtttatt aaagtgttaa
     1981 ttcacagctt tattgcttgt tttgagaatc tgcttgcata agtgattttt aaacttgatc
     2041 tgattttcgt ttgtttcgaa gttcttcagc tttgaaagtc agatcttcaa ttcacagcca
     2101 tgccatagtt ttaggataca gtataagtag acgaattggg atgcatcgta gattttatgc
     2161 tgtgtttgtt atggtttttc agtggtagag attttcagag tattattagg gttatagtca
     2221 gtgtcaggtt tatttatcgt cagcgtgatg ggcaaaaaac agctggcagt acgggctgga
     2281 acggacaatg gatgcctggc tccatcttac aaggcagttt tggatgttat atcttctcta
     2341 atactgcctg gtaatgatgc agatggtcat ctagaggcaa ctttgcatct gggtgtggac
     2401 atgctgacta tgcaaatatg catacaaatg gaaaatatgc catggtgaaa tgtgttctca
     2461 ggtcctgtga tgctgaagga gttcattcat tgtctctagg gtacatctta cctgacagtg
     2521 cttggctgtt cactgatgtt ctaacactgt ctggtaacga tgcactctgg tgactcagcc
     2581 aactgctgtt actctcgccc agaaatctgc gccaacatgg acacacccag tggagcaagc
     2641 catctgattt tcatggtcag attgtaaaga agcaattact cttggacagt ttaagcagaa
     2701 aagccaccaa gtcaagtcac tggtcatatg atgaactcga agatctaata aaagttaatt
     2761 taagcgttca ttacaataaa ttcaagtgaa tctgttttaa gctagccttt aacgttttta
     2821 aattccttca aacaaattag ttcctctttg cagttcctca tttctcggtg agctggtgct
     2881 ttaatccctt tactttttct tcctctaggg agtgatggtt ggcatgggac gaattaattc
     2941 ctgcagcccg ggggtaccgg tcgccaccat gaacaccccg ggaattaacc tgatcaagga
     3001 ggacatgcgc gtgaaggtgc acatggaggg caacgtgaac ggccacgcct tcgtgatcga
     3061 gggcgagggc aagggcaagc cctacgaggg cacccagacc gccaacctga ccgtgaagga
     3121 gggcgccccc ctgcccttca gctacgacat cctgaccacc gccgtgcact acggcaaccg
     3181 ggtgttcacc aagtaccccg aggacatccc cgactacttc aagcagagct tccccgaggg
     3241 ctacagctgg gagcgcacca tgaccttcga ggacaagggc atctgcacca tccgcagcga
     3301 catcagcctg gagggcgact gcttcttcca gaacgtgcgc ttcaagggca ccaacttccc
     3361 ccccaacggc cccgtgatgc agaagaaaac cctgaagtgg gagcccagca ccgagaagct
     3421 gcacgtgcgc gacggcctgc tggtgggcaa catcaacatg gccctgctgc tggagggcgg
     3481 cggccactac ctgtgcgact tcaagaccac ctacaaggcc aagaaggtgg tgcagctgcc
     3541 cgacgcccac ttcgtggacc accgcatcga gatcctgggc aacgacagcg actacaacaa
     3601 ggtgaagctg tacgagcacg ccgtggcccg ctacagcccc ctgcccagcc aggtgtggta
     3661 atctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3721 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     3781 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3841 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     3901 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3961 ttttaaccaa taggccgaaa tcggcggatc ctgcggccgc aagggaaaat agaatgaagt
     4021 gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact
     4081 tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca
     4141 agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa
     4201 gtactttaca cctctggttc ttgaccccct accttcagca agcccagcag atccactaga
     4261 tcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     4321 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4381 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4441 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4501 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4561 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4621 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4681 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4741 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4801 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4861 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4921 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4981 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5041 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5101 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5161 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5221 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5281 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5341 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5401 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5461 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5521 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5581 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5641 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5701 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5761 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5821 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5881 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5941 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6001 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6061 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6121 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6181 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6241 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6301 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6361 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6421 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     6481 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     6541 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     6601 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6661 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6721 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6781 gggaaagccg gccacaatga cggctactac atggtgccat tccttcctct ttataggaat
     6841 ggagactacc tcctgtccaa caatgctctt ggatacgagt acgcctacct gttggaccca
     6901 ggtcattgca caacaccaga aatgccctct gatctgcaaa agacgtgaat atctgttcag
     6961 acacccatat ccactctgtt ccacacaggt cagaggtttg tccaggagtt cttgacagag
     7021 gtgtaaaaag tactcaaaaa ttttactcaa gtgaaagtac aagtacttag ggaaaatttt
     7081 actcaattaa aagtaaaagt atctggctag aatcttactt gagtaaaagt aaaaaagtac
     7141 tccattaaaa ttgtacttga gtattaagga agtaaaaggc tagcaagatc tgctcgagat
     7201 acatttgatt agaagagttt gacttgggtt gattatagca aatagtggaa cacaaacaga
     7261 ttttgaaata ttcttgcaaa tttatttatt aaatccattc aaatattgtc attttttgta
     7321 ctgtgtaatt tatattgatt tgctcataca atatttacat ttcgcttaaa caatacacta
     7381 ttatttatat ttaaaaatag ctatatataa atcaaatgta tagctctttt ttacacaatt
     7441 aatcaaagct aaaatatttg tcacacaaat acagtaagcc acttcacaat gatatcttca
     7501 aaatctgtgc ttgactgaat catttcattt attgttttgt aattcacatt ataccttacg
     7561 ttctccaggc acagtttgaa ttgttgataa atgaagataa tacatctgat ttaattagtc
     7621 tcctttgttt ataataactt taatctaatt tgatagtaat aattccagca atacatccat
     7681 tacaataaaa atcgcttttt tattttgcac attagtttgc cggtatttgt ttaaagtttc
     7741 accatacttt taaatcatgt gctatttgaa aagggctgtt atgactggaa ttaaatgttt
     7801 ttttttatat cagtgtttta gattgaagta caatatttat aggtaaaatt caccagcata
     7861 tgcttaaaga ta
//
