LOCUS       sequence-182643-        7871 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 7871)
  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 7871)
  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 184-1-dendra2
FEATURES             Location/Qualifiers
     source          1..7871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1698..1707
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2945..3637
                     /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    3748..3869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3785..3804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3839..3858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3993..4180,4181..4192)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4247..4267)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4247..4265)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4286..4302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4286..4302)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4299..4321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4310..4326
                     /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(4334..4340,4341..4358,4359..4364))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4379..4400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4517..4534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4688..5276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4768..4787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5447..6238,6239..6307))
                     /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     6070..6089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6308..6412)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6575..6596)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6992..7003,7004..7141)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 cttctaaggt ctgtttgtgg tttaagctgt ttgttgacac acgcttgtca tctccatttt
       61 tctgtgaagt tattgtcagt ttattgcaca ctgggttgca ttaagctgca gttttttcac
      121 aacagttttc cacattgtaa cagtcagtct ccaaattatt ccatcagtcc tcaaatttag
      181 tcatttgagg ccatgaaaac acttaaaact acatgaaggt agtttggcat taggttttag
      241 acttgttctt gtttggcata ttgcaacctg tgtaaaaatg atttgatttt tatttttttt
      301 tttttttttt cagaaagaaa cggtccaaaa agagttatgt gaacatttat ccatgtgttt
      361 aatgtttttt aaattaaaaa aaaacatttt tattaaacca cttgtgacta aacctttttt
      421 agttattcta ttttgtgacg agtctgaatg gccaaagtac tcttgaatcc aaaatcttta
      481 tagtaaaatg tttaatctta cttgcagtgt tgggtataat ttgtaacaaa gttgaattcc
      541 atatctgctg agtgaaaaag taaagtaaga gattgctgta aatatttagg ttatttattt
      601 attaatacaa tatagaaagt aacaaaacac aatgtaaaaa atcaataaag aaagtaaata
      661 ttaaaagtaa tgataaacta atgatgtaaa caatattagc aacaaagttt gtacaattca
      721 gggagggtaa agaatttcac ctaatagtca aactctgtat taaataatat aaaatctttg
      781 ttcttagtct gaagaggaga ctgtgagaga aagcttactg gttgttaaat tagaaaattt
      841 gtaacttgca gattaaaatg atttaaataa ttttataaag gccatgtttg aaacaaatgt
      901 aaaaacataa taaatgttaa gaatttcaga tattgagatt ttacaattta aaaaaagcaa
      961 taaagtaata gcagagacct acactcttaa agtaaattac ccaactctgc ttactagtaa
     1021 cttttggaat agttactagt atccattgaa caaatactag aatctaatta aattgtgtga
     1081 gtgtccaatg aaaagattct tgtatttaaa acagtaccag tatctatata tgtacaaaat
     1141 ttagtgctag caaaatgcaa ataaatacta ctgcatataa tggattgcat tgtaaatcga
     1201 ctcgccataa tacatttatt tcctattttt acttttaaca aaaaataagt tcactgagta
     1261 tttcatgtca ttaaatcgca ttaaacctct tgcatgcttt atgtacacta gtgcagttcg
     1321 ctaaatgacc accggtgtca ctaataacac gatcgtttct gaattctgca ttaaacgcca
     1381 tttatacgtt gctcttatta gtaaacaatt tctccatcat gagctcgacc agtattgcat
     1441 atttcatcct cttaaatcca gagttgtttc ttttaatcac tttctcttca tgataaaagc
     1501 agtttttggt tttcagaagt ttacaacaaa cccagtcagt tattgcctca gcaaacactg
     1561 gagcaactgc acacttgtga aaagcttctt gcgaaacaca aacctttctc tggacttata
     1621 aataaaggca ttcagcagct ccaccataca cctccactgg tcttgataaa gcagatatca
     1681 gcagtgatac aggtaccgcc accatggatg aggaaatcgc tgccctggtc gttgacaacg
     1741 gctccggtat gtgcaaagcc ggttttgctg gagatgatgc ccctcgtgct gttttcccct
     1801 ccattgttgg acgacccaga catcaggtac atttaataaa ccatcctaat aacatgttaa
     1861 tcatattgaa ccacattaaa ctctcatttg tgcattttac atcttttaaa accgatttat
     1921 attcgttttt agataatgaa gtttattaaa gtgttaattc acagctttat tgcttgtttt
     1981 gagaatctgc ttgcataagt gatttttaaa cttgatctga ttttcgtttg tttcgaagtt
     2041 cttcagcttt gaaagtcaga tcttcaattc acagccatgc catagtttta ggatacagta
     2101 taagtagacg aattgggatg catcgtagat tttatgctgt gtttgttatg gtttttcagt
     2161 ggtagagatt ttcagagtat tattagggtt atagtcagtg tcaggtttat ttatcgtcag
     2221 cgtgatgggc aaaaaacagc tggcagtacg ggcttcagca aaggcgttca cactaataat
     2281 tatagaaatg atcaaaggat atagacccag atggactggc agggagtatc gggcacatgc
     2341 ccttatcagt tctccgtcca acgaatacta aatggatagc tgggctggaa aagtgataag
     2401 gagacgtgtt cgactgaaat caaattctca cccttatgga agaatccgct gtgtgttttg
     2461 tatgtccaac tatccacagt gagacatgta gacatccaaa ataatggcat cagaagaatg
     2521 tcaatgtcca aatggctcat ctccgacaaa cgagccaact gctgttactc tcgcccagaa
     2581 atctgcgcca acatggacac acccagtgga gcaagccatc tgattttcat ggtcagattg
     2641 taaagaagca attactcttg gacagtttaa gcagaaaagc caccaagtca agtcactggt
     2701 catatgatga actcgaagat ctaataaaag ttaatttaag cgttcattac aataaattca
     2761 agtgaatctg ttttaagcta gcctttaacg tttttaaatt ccttcaaaca aattagttcc
     2821 tctttgcagt tcctcatttc tcggtgagct ggtgctttaa tccctttact ttttcttcct
     2881 ctagggagtg atggttggca tgggacgaat taattcctgc agcccggggg taccggtcgc
     2941 caccatgaac accccgggaa ttaacctgat caaggaggac atgcgcgtga aggtgcacat
     3001 ggagggcaac gtgaacggcc acgccttcgt gatcgagggc gagggcaagg gcaagcccta
     3061 cgagggcacc cagaccgcca acctgaccgt gaaggagggc gcccccctgc ccttcagcta
     3121 cgacatcctg accaccgccg tgcactacgg caaccgggtg ttcaccaagt accccgagga
     3181 catccccgac tacttcaagc agagcttccc cgagggctac agctgggagc gcaccatgac
     3241 cttcgaggac aagggcatct gcaccatccg cagcgacatc agcctggagg gcgactgctt
     3301 cttccagaac gtgcgcttca agggcaccaa cttccccccc aacggccccg tgatgcagaa
     3361 gaaaaccctg aagtgggagc ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt
     3421 gggcaacatc aacatggccc tgctgctgga gggcggcggc cactacctgt gcgacttcaa
     3481 gaccacctac aaggccaaga aggtggtgca gctgcccgac gcccacttcg tggaccaccg
     3541 catcgagatc ctgggcaacg acagcgacta caacaaggtg aagctgtacg agcacgccgt
     3601 ggcccgctac agccccctgc ccagccaggt gtggtaatct agatcataat cagccatacc
     3661 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     3721 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     3781 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3841 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     3901 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3961 cggatcctgc ggccgcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt
     4021 tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta
     4081 agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat
     4141 aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga
     4201 ccccctacct tcagcaagcc cagcagatcc actagatctt ttgttccctt tagtgagggt
     4261 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4321 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4381 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4441 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4501 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4561 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4621 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4681 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4741 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4801 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4861 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4921 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4981 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5041 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5101 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5161 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5221 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5281 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5341 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5401 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5461 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5521 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5581 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5641 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5701 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5761 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5821 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5881 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5941 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6001 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6061 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6121 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6181 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6241 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6301 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6361 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6421 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     6481 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6541 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6601 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6661 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6721 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcca caatgacggc
     6781 tactacatgg tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat
     6841 gctcttggat acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg
     6901 ccctctgatc tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac
     6961 acaggtcaga ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt
     7021 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct
     7081 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat
     7141 taaggaagta aaaggctagc aagatctgct cgagatacat ttgattagaa gagtttgact
     7201 tgggttgatt atagcaaata gtggaacaca aacagatttt gaaatattct tgcaaattta
     7261 tttattaaat ccattcaaat attgtcattt tttgtactgt gtaatttata ttgatttgct
     7321 catacaatat ttacatttcg cttaaacaat acactattat ttatatttaa aaatagctat
     7381 atataaatca aatgtatagc tcttttttac acaattaatc aaagctaaaa tatttgtcac
     7441 acaaatacag taagccactt cacaatgata tcttcaaaat ctgtgcttga ctgaatcatt
     7501 tcatttattg ttttgtaatt cacattatac cttacgttct ccaggcacag tttgaattgt
     7561 tgataaatga agataataca tctgatttaa ttagtctcct ttgtttataa taactttaat
     7621 ctaatttgat agtaataatt ccagcaatac atccattaca ataaaaatcg cttttttatt
     7681 ttgcacatta gtttgccggt atttgtttaa agtttcacca tacttttaaa tcatgtgcta
     7741 tttgaaaagg gctgttatga ctggaattaa atgttttttt ttatatcagt gttttagatt
     7801 gaagtacaat atttataggt aaaattcacc agcatatgct taaagatagc catgctccag
     7861 gtccttgtgt c
//
