LOCUS       sequence-182775-        7994 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 7994)
  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 7994)
  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 725-Dendra2
FEATURES             Location/Qualifiers
     source          1..7994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1715..1724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3085..3777
                     /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    3888..4009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3925..3944)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3979..3998
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4133..4320,4321..4332)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4387..4407)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4387..4405)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4426..4442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4426..4442)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4439..4461)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4450..4466
                     /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(4474..4480,4481..4498,4499..4504))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4519..4540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4657..4674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4828..5416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4908..4927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5587..6378,6379..6447))
                     /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     6210..6229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6448..6552)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6715..6736)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7132..7143,7144..7281)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ctccaggtcc ttgtgtcctt ctaaggtctg tttgtggttt aagctgtttg ttgacacacg
       61 cttgtcatct ccatttttct gtgaagttat tgtcagttta ttgcacactg ggttgcatta
      121 agctgcagtt ttttcacaac agttttccac attgtaacag tcagtctcca aattattcca
      181 tcagtcctca aatttagtca tttgaggcca tgaaaacact taaaactaca tgaaggtagt
      241 ttggcattag gttttagact tgttcttgtt tggcatattg caacctgtgt aaaaatgatt
      301 tgatttttat tttttttttt tttttttcag aaagaaacgg tccaaaaaga gttatgtgaa
      361 catttatcca tgtgtttaat gttttttaaa ttaaaaaaaa acatttttat taaaccactt
      421 gtgactaaac cttttttagt tattctattt tgtgacgagt ctgaatggcc aaagtactct
      481 tgaatccaaa atctttatag taaaatgttt aatcttactt gcagtgttgg gtataatttg
      541 taacaaagtt gaattccata tctgctgagt gaaaaagtaa agtaagagat tgctgtaaat
      601 atttaggtta tttatttatt aatacaatat agaaagtaac aaaacacaat gtaaaaaatc
      661 aataaagaaa gtaaatatta aaagtaatga taaactaatg atgtaaacaa tattagcaac
      721 aaagtttgta caattcaggg agggtaaaga atttcaccta atagtcaaac tctgtattaa
      781 ataatataaa atctttgttc ttagtctgaa gaggagactg tgagagaaag cttactggtt
      841 gttaaattag aaaatttgta acttgcagat taaaatgatt taaataattt tataaaggcc
      901 atgtttgaaa caaatgtaaa aacataataa atgttaagaa tttcagatat tgagatttta
      961 caatttaaaa aaagcaataa agtaatagca gagacctaca ctcttaaagt aaattaccca
     1021 actctgctta ctagtaactt ttggaatagt tactagtatc cattgaacaa atactagaat
     1081 ctaattaaat tgtgtgagtg tccaatgaaa agattcttgt atttaaaaca gtaccagtat
     1141 ctatatatgt acaaaattta gtgctagcaa aatgcaaata aatactactg catataatgg
     1201 attgcattgt aaatcgactc gccataatac atttatttcc tatttttact tttaacaaaa
     1261 aataagttca ctgagtattt catgtcatta aatcgcatta aacctcttgc atgctttatg
     1321 tacactagtg cagttcgcta aatgaccacc ggtgtcacta ataacacgat cgtttctgaa
     1381 ttctgcatta aacgccattt atacgttgct cttattagta aacaatttct ccatcatgag
     1441 ctcgaccagt attgcatatt tcatcctctt aaatccagag ttgtttcttt taatcacttt
     1501 ctcttcatga taaaagcagt ttttggtttt cagaagttta caacaaaccc agtcagttat
     1561 tgcctcagca aacactggag caactgcaca cttgtgaaaa gcttcttgcg aaacacaaac
     1621 ctttctctgg acttataaat aaaggcattc agcagctcca ccatacacct ccactggtct
     1681 tgataaagca gatatcagca gtgatacagg taccgccacc atggatgagg aaatcgctgc
     1741 cctggtcgtt gacaacggct ccggtatgtg caaagccggt tttgctggag atgatgcccc
     1801 tcgtgctgtt ttcccctcca ttgttggacg acccagacat caggtacatt taataaacca
     1861 tcctaataac atgttaatca tattgaacca cattaaactc tcatttgtgc attttacatc
     1921 ttttaaaacc gatttatatt cgtttttaga taatgaagtt tattaaagtg ttaattcaca
     1981 gctttattgc ttgttttgag aatctgcttg cataagtgat ttttaaactt gatctgattt
     2041 tcgtttgttt cgaagttctt cagctttgaa agtcagatct tcaattcaca gccatgccat
     2101 agttttagga tacagtataa gtagacgaat tgggatgcat cgtagatttt atgctgtgtt
     2161 tgttatggtt tttcagtggt agagattttc agagtattat tagggttata gtcagtgtca
     2221 ggtttattta tcgtcagcgt gatgggcaaa aaacagctgg cagtacgggc tgttgcattt
     2281 taccgtgaca cccattttaa gtgcaaacat tcaatgtaat aataataata atgccaggat
     2341 tttttaagac acactgggaa attgttgaaa aacattaagc aatgttgaaa taaatttaag
     2401 tagattaaga taagtggtta aagtataata tctacctaat aataaaaata ataattatta
     2461 ttattattaa aaacgaagtt attttaaagt ggaacatgtg catgaaattt catactgcca
     2521 tttttattcc agatgttgca taatacatgt ttacctggac gtaagacctg cagtgcacat
     2581 tgctaggaat ggtggctgag atgaagagca ggatttcagt cattgtttct agtagtgcgc
     2641 gctgcagata ttcagctttc ctaaactcaa atccgcaaac ctgagaagag gaagccaact
     2701 gctgttactc tcgcccagaa atctgcgcca acatggacac acccagtgga gcaagccatc
     2761 tgattttcat ggtcagattg taaagaagca attactcttg gacagtttaa gcagaaaagc
     2821 caccaagtca agtcactggt catatgatga actcgaagat ctaataaaag ttaatttaag
     2881 cgttcattac aataaattca agtgaatctg ttttaagcta gcctttaacg tttttaaatt
     2941 ccttcaaaca aattagttcc tctttgcagt tcctcatttc tcggtgagct ggtgctttaa
     3001 tccctttact ttttcttcct ctagggagtg atggttggca tgggacgaat taattcctgc
     3061 agcccggggg taccggtcgc caccatgaac accccgggaa ttaacctgat caaggaggac
     3121 atgcgcgtga aggtgcacat ggagggcaac gtgaacggcc acgccttcgt gatcgagggc
     3181 gagggcaagg gcaagcccta cgagggcacc cagaccgcca acctgaccgt gaaggagggc
     3241 gcccccctgc ccttcagcta cgacatcctg accaccgccg tgcactacgg caaccgggtg
     3301 ttcaccaagt accccgagga catccccgac tacttcaagc agagcttccc cgagggctac
     3361 agctgggagc gcaccatgac cttcgaggac aagggcatct gcaccatccg cagcgacatc
     3421 agcctggagg gcgactgctt cttccagaac gtgcgcttca agggcaccaa cttccccccc
     3481 aacggccccg tgatgcagaa gaaaaccctg aagtgggagc ccagcaccga gaagctgcac
     3541 gtgcgcgacg gcctgctggt gggcaacatc aacatggccc tgctgctgga gggcggcggc
     3601 cactacctgt gcgacttcaa gaccacctac aaggccaaga aggtggtgca gctgcccgac
     3661 gcccacttcg tggaccaccg catcgagatc ctgggcaacg acagcgacta caacaaggtg
     3721 aagctgtacg agcacgccgt ggcccgctac agccccctgc ccagccaggt gtggtaatct
     3781 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3841 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3901 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3961 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     4021 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4081 aaccaatagg ccgaaatcgg cggatcctgc ggccgcaagg gaaaatagaa tgaagtgatc
     4141 tccaaaaaat aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt
     4201 tttctaaaaa aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta
     4261 aagtaaaaat ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac
     4321 tttacacctc tggttcttga ccccctacct tcagcaagcc cagcagatcc actagatctt
     4381 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4441 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4501 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4561 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4621 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4681 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4741 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4801 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4861 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4921 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4981 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5041 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5101 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5161 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5221 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5281 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5341 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5401 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5461 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5521 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5581 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5641 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5701 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5761 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5821 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5881 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5941 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6001 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6061 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6121 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6181 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6241 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6301 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6361 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6421 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6481 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6541 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     6601 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     6661 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     6721 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6781 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6841 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6901 aagccggcca caatgacggc tactacatgg tgccattcct tcctctttat aggaatggag
     6961 actacctcct gtccaacaat gctcttggat acgagtacgc ctacctgttg gacccaggtc
     7021 attgcacaac accagaaatg ccctctgatc tgcaaaagac gtgaatatct gttcagacac
     7081 ccatatccac tctgttccac acaggtcaga ggtttgtcca ggagttcttg acagaggtgt
     7141 aaaaagtact caaaaatttt actcaagtga aagtacaagt acttagggaa aattttactc
     7201 aattaaaagt aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa aagtactcca
     7261 ttaaaattgt acttgagtat taaggaagta aaaggctagc aagatctgct cgagatacat
     7321 ttgattagaa gagtttgact tgggttgatt atagcaaata gtggaacaca aacagatttt
     7381 gaaatattct tgcaaattta tttattaaat ccattcaaat attgtcattt tttgtactgt
     7441 gtaatttata ttgatttgct catacaatat ttacatttcg cttaaacaat acactattat
     7501 ttatatttaa aaatagctat atataaatca aatgtatagc tcttttttac acaattaatc
     7561 aaagctaaaa tatttgtcac acaaatacag taagccactt cacaatgata tcttcaaaat
     7621 ctgtgcttga ctgaatcatt tcatttattg ttttgtaatt cacattatac cttacgttct
     7681 ccaggcacag tttgaattgt tgataaatga agataataca tctgatttaa ttagtctcct
     7741 ttgtttataa taactttaat ctaatttgat agtaataatt ccagcaatac atccattaca
     7801 ataaaaatcg cttttttatt ttgcacatta gtttgccggt atttgtttaa agtttcacca
     7861 tacttttaaa tcatgtgcta tttgaaaagg gctgttatga ctggaattaa atgttttttt
     7921 ttatatcagt gttttagatt gaagtacaat atttataggt aaaattcacc agcatatgct
     7981 taaagatagc catg
//
