LOCUS       sequence-182641-        7850 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 7850)
  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 7850)
  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 182-dendra2
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1713..1722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2939..3631
                     /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    3742..3863
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3779..3798)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3833..3852
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3987..4174,4175..4186)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4241..4261)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4241..4259)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4280..4296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4280..4296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4293..4315)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4304..4320
                     /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(4328..4334,4335..4352,4353..4358))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4373..4394
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4511..4528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4682..5270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4762..4781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5441..6232,6233..6301))
                     /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     6064..6083
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6302..6406)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6569..6590)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6986..6997,6998..7135)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ccaggtcctt gtgtccttct aaggtctgtt tgtggtttaa gctgtttgtt gacacacgct
       61 tgtcatctcc atttttctgt gaagttattg tcagtttatt gcacactggg ttgcattaag
      121 ctgcagtttt ttcacaacag ttttccacat tgtaacagtc agtctccaaa ttattccatc
      181 agtcctcaaa tttagtcatt tgaggccatg aaaacactta aaactacatg aaggtagttt
      241 ggcattaggt tttagacttg ttcttgtttg gcatattgca acctgtgtaa aaatgatttg
      301 atttttattt tttttttttt tttttcagaa agaaacggtc caaaaagagt tatgtgaaca
      361 tttatccatg tgtttaatgt tttttaaatt aaaaaaaaac atttttatta aaccacttgt
      421 gactaaacct tttttagtta ttctattttg tgacgagtct gaatggccaa agtactcttg
      481 aatccaaaat ctttatagta aaatgtttaa tcttacttgc agtgttgggt ataatttgta
      541 acaaagttga attccatatc tgctgagtga aaaagtaaag taagagattg ctgtaaatat
      601 ttaggttatt tatttattaa tacaatatag aaagtaacaa aacacaatgt aaaaaatcaa
      661 taaagaaagt aaatattaaa agtaatgata aactaatgat gtaaacaata ttagcaacaa
      721 agtttgtaca attcagggag ggtaaagaat ttcacctaat agtcaaactc tgtattaaat
      781 aatataaaat ctttgttctt agtctgaaga ggagactgtg agagaaagct tactggttgt
      841 taaattagaa aatttgtaac ttgcagatta aaatgattta aataatttta taaaggccat
      901 gtttgaaaca aatgtaaaaa cataataaat gttaagaatt tcagatattg agattttaca
      961 atttaaaaaa agcaataaag taatagcaga gacctacact cttaaagtaa attacccaac
     1021 tctgcttact agtaactttt ggaatagtta ctagtatcca ttgaacaaat actagaatct
     1081 aattaaattg tgtgagtgtc caatgaaaag attcttgtat ttaaaacagt accagtatct
     1141 atatatgtac aaaatttagt gctagcaaaa tgcaaataaa tactactgca tataatggat
     1201 tgcattgtaa atcgactcgc cataatacat ttatttccta tttttacttt taacaaaaaa
     1261 taagttcact gagtatttca tgtcattaaa tcgcattaaa cctcttgcat gctttatgta
     1321 cactagtgca gttcgctaaa tgaccaccgg tgtcactaat aacacgatcg tttctgaatt
     1381 ctgcattaaa cgccatttat acgttgctct tattagtaaa caatttctcc atcatgagct
     1441 cgaccagtat tgcatatttc atcctcttaa atccagagtt gtttctttta atcactttct
     1501 cttcatgata aaagcagttt ttggttttca gaagtttaca acaaacccag tcagttattg
     1561 cctcagcaaa cactggagca actgcacact tgtgaaaagc ttcttgcgaa acacaaacct
     1621 ttctctggac ttataaataa aggcattcag cagctccacc atacacctcc actggtcttg
     1681 ataaagcaga tatcagcagt gatacaggta ccgccaccat ggatgaggaa atcgctgccc
     1741 tggtcgttga caacggctcc ggtatgtgca aagccggttt tgctggagat gatgcccctc
     1801 gtgctgtttt cccctccatt gttggacgac ccagacatca ggtacattta ataaaccatc
     1861 ctaataacat gttaatcata ttgaaccaca ttaaactctc atttgtgcat tttacatctt
     1921 ttaaaaccga tttatattcg tttttagata atgaagttta ttaaagtgtt aattcacagc
     1981 tttattgctt gttttgagaa tctgcttgca taagtgattt ttaaacttga tctgattttc
     2041 gtttgtttcg aagttcttca gctttgaaag tcagatcttc aattcacagc catgccatag
     2101 ttttaggata cagtataagt agacgaattg ggatgcatcg tagattttat gctgtgtttg
     2161 ttatggtttt tcagtggtag agattttcag agtattatta gggttatagt cagtgtcagg
     2221 tttatttatc gtcagcgtga tgggcaaaaa acagctggca gtacgggctc gatgtctcaa
     2281 tgaatggccc atttcttcca ctactatttc aaggacaaaa ggttctctga tggtatttgg
     2341 caatggtaga actcacactg gtgaggtagt cagatccggt ggttctagac ttgccaacta
     2401 ctacctgaga acaactccac tgtaaggcat ggatataaac aaatatagac aaagaactca
     2461 gagcataatt tcgaatatca aggttgtttc aatgatgtca caagctccgc cccctgagcg
     2521 agtgactgtg agtagtgcag gttgggagcc aactgctgtt actctcgccc agaaatctgc
     2581 gccaacatgg acacacccag tggagcaagc catctgattt tcatggtcag attgtaaaga
     2641 agcaattact cttggacagt ttaagcagaa aagccaccaa gtcaagtcac tggtcatatg
     2701 atgaactcga agatctaata aaagttaatt taagcgttca ttacaataaa ttcaagtgaa
     2761 tctgttttaa gctagccttt aacgttttta aattccttca aacaaattag ttcctctttg
     2821 cagttcctca tttctcggtg agctggtgct ttaatccctt tactttttct tcctctaggg
     2881 agtgatggtt ggcatgggac gaattaattc ctgcagcccg ggggtaccgg tcgccaccat
     2941 gaacaccccg ggaattaacc tgatcaagga ggacatgcgc gtgaaggtgc acatggaggg
     3001 caacgtgaac ggccacgcct tcgtgatcga gggcgagggc aagggcaagc cctacgaggg
     3061 cacccagacc gccaacctga ccgtgaagga gggcgccccc ctgcccttca gctacgacat
     3121 cctgaccacc gccgtgcact acggcaaccg ggtgttcacc aagtaccccg aggacatccc
     3181 cgactacttc aagcagagct tccccgaggg ctacagctgg gagcgcacca tgaccttcga
     3241 ggacaagggc atctgcacca tccgcagcga catcagcctg gagggcgact gcttcttcca
     3301 gaacgtgcgc ttcaagggca ccaacttccc ccccaacggc cccgtgatgc agaagaaaac
     3361 cctgaagtgg gagcccagca ccgagaagct gcacgtgcgc gacggcctgc tggtgggcaa
     3421 catcaacatg gccctgctgc tggagggcgg cggccactac ctgtgcgact tcaagaccac
     3481 ctacaaggcc aagaaggtgg tgcagctgcc cgacgcccac ttcgtggacc accgcatcga
     3541 gatcctgggc aacgacagcg actacaacaa ggtgaagctg tacgagcacg ccgtggcccg
     3601 ctacagcccc ctgcccagcc aggtgtggta atctagatca taatcagcca taccacattt
     3661 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     3721 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     3781 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3841 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     3901 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcggatc
     3961 ctgcggccgc aagggaaaat agaatgaagt gatctccaaa aaataagtac tttttgactg
     4021 taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta attaagtaaa
     4081 agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa aaataatact
     4141 taagtacagt aatcaagtaa aattactcaa gtactttaca cctctggttc ttgaccccct
     4201 accttcagca agcccagcag atccactaga tcttttgttc cctttagtga gggttaattg
     4261 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4321 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4381 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4441 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4501 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4561 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4621 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4681 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4741 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4801 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4861 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4921 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4981 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5041 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5101 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5161 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5221 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5281 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5341 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5401 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5461 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5521 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5581 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5641 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5701 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5761 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5821 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5881 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5941 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6001 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6061 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6121 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6181 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6241 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6301 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6361 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6421 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     6481 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     6541 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6601 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6661 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6721 taaagggagc ccccgattta gagcttgacg gggaaagccg gccacaatga cggctactac
     6781 atggtgccat tccttcctct ttataggaat ggagactacc tcctgtccaa caatgctctt
     6841 ggatacgagt acgcctacct gttggaccca ggtcattgca caacaccaga aatgccctct
     6901 gatctgcaaa agacgtgaat atctgttcag acacccatat ccactctgtt ccacacaggt
     6961 cagaggtttg tccaggagtt cttgacagag gtgtaaaaag tactcaaaaa ttttactcaa
     7021 gtgaaagtac aagtacttag ggaaaatttt actcaattaa aagtaaaagt atctggctag
     7081 aatcttactt gagtaaaagt aaaaaagtac tccattaaaa ttgtacttga gtattaagga
     7141 agtaaaaggc tagcaagatc tgctcgagat acatttgatt agaagagttt gacttgggtt
     7201 gattatagca aatagtggaa cacaaacaga ttttgaaata ttcttgcaaa tttatttatt
     7261 aaatccattc aaatattgtc attttttgta ctgtgtaatt tatattgatt tgctcataca
     7321 atatttacat ttcgcttaaa caatacacta ttatttatat ttaaaaatag ctatatataa
     7381 atcaaatgta tagctctttt ttacacaatt aatcaaagct aaaatatttg tcacacaaat
     7441 acagtaagcc acttcacaat gatatcttca aaatctgtgc ttgactgaat catttcattt
     7501 attgttttgt aattcacatt ataccttacg ttctccaggc acagtttgaa ttgttgataa
     7561 atgaagataa tacatctgat ttaattagtc tcctttgttt ataataactt taatctaatt
     7621 tgatagtaat aattccagca atacatccat tacaataaaa atcgcttttt tattttgcac
     7681 attagtttgc cggtatttgt ttaaagtttc accatacttt taaatcatgt gctatttgaa
     7741 aagggctgtt atgactggaa ttaaatgttt ttttttatat cagtgtttta gattgaagta
     7801 caatatttat aggtaaaatt caccagcata tgcttaaaga tagccatgct
//
