LOCUS       sequence-182596-        8015 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 8015)
  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 8015)
  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 101a-Dendra2
FEATURES             Location/Qualifiers
     source          1..8015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1787..1796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3172..3864
                     /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    3975..4096
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4012..4031)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4066..4085
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4480..4500)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4480..4498)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4519..4535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4519..4535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4532..4554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4543..4559
                     /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(4567..4573,4574..4591,4592..4597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4612..4633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4750..4767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4921..5509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5001..5020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5680..6471,6472..6540))
                     /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     6303..6322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6541..6645)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6808..6829)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7225..7236,7237..7374)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 atatcagtgt tttagattga agtacaatat ttataggtaa aattcaccag catatgctta
       61 aagatagcca tgctccaggt ccttgtgtcc ttctaaggtc tgtttgtggt ttaagctgtt
      121 tgttgacaca cgcttgtcat ctccattttt ctgtgaagtt attgtcagtt tattgcacac
      181 tgggttgcat taagctgcag ttttttcaca acagttttcc acattgtaac agtcagtctc
      241 caaattattc catcagtcct caaatttagt catttgaggc catgaaaaca cttaaaacta
      301 catgaaggta gtttggcatt aggttttaga cttgttcttg tttggcatat tgcaacctgt
      361 gtaaaaatga tttgattttt attttttttt tttttttttc agaaagaaac ggtccaaaaa
      421 gagttatgtg aacatttatc catgtgttta atgtttttta aattaaaaaa aaacattttt
      481 attaaaccac ttgtgactaa acctttttta gttattctat tttgtgacga gtctgaatgg
      541 ccaaagtact cttgaatcca aaatctttat agtaaaatgt ttaatcttac ttgcagtgtt
      601 gggtataatt tgtaacaaag ttgaattcca tatctgctga gtgaaaaagt aaagtaagag
      661 attgctgtaa atatttaggt tatttattta ttaatacaat atagaaagta acaaaacaca
      721 atgtaaaaaa tcaataaaga aagtaaatat taaaagtaat gataaactaa tgatgtaaac
      781 aatattagca acaaagtttg tacaattcag ggagggtaaa gaatttcacc taatagtcaa
      841 actctgtatt aaataatata aaatctttgt tcttagtctg aagaggagac tgtgagagaa
      901 agcttactgg ttgttaaatt agaaaatttg taacttgcag attaaaatga tttaaataat
      961 tttataaagg ccatgtttga aacaaatgta aaaacataat aaatgttaag aatttcagat
     1021 attgagattt tacaatttaa aaaaagcaat aaagtaatag cagagaccta cactcttaaa
     1081 gtaaattacc caactctgct tactagtaac ttttggaata gttactagta tccattgaac
     1141 aaatactaga atctaattaa attgtgtgag tgtccaatga aaagattctt gtatttaaaa
     1201 cagtaccagt atctatatat gtacaaaatt tagtgctagc aaaatgcaaa taaatactac
     1261 tgcatataat ggattgcatt gtaaatcgac tcgccataat acatttattt cctattttta
     1321 cttttaacaa aaaataagtt cactgagtat ttcatgtcat taaatcgcat taaacctctt
     1381 gcatgcttta tgtacactag tgcagttcgc taaatgacca ccggtgtcac taataacacg
     1441 atcgtttctg aattctgcat taaacgccat ttatacgttg ctcttattag taaacaattt
     1501 ctccatcatg agctcgacca gtattgcata tttcatcctc ttaaatccag agttgtttct
     1561 tttaatcact ttctcttcat gataaaagca gtttttggtt ttcagaagtt tacaacaaac
     1621 ccagtcagtt attgcctcag caaacactgg agcaactgca cacttgtgaa aagcttcttg
     1681 cgaaacacaa acctttctct ggacttataa ataaaggcat tcagcagctc caccatacac
     1741 ctccactggt cttgataaag cagatatcag cagtgataca ggtaccgcca ccatggatga
     1801 ggaaatcgct gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg
     1861 agatgatgcc cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca
     1921 tttaataaac catcctaata acatgttaat catattgaac cacattaaac tctcatttgt
     1981 gcattttaca tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag
     2041 tgttaattca cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac
     2101 ttgatctgat tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca
     2161 cagccatgcc atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt
     2221 ttatgctgtg tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta
     2281 tagtcagtgt caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg
     2341 gctgagaggg tgctgtaact tatccaataa aaatatgcaa tgtagcccaa tcatgacatt
     2401 atagatgaac tggatggtaa aagtttgttt tgtcatagaa ttcttcattg agtgcatcta
     2461 ctcatgtcct taatctattt gcagatgcat taatttgctt cattttttat attctgcaat
     2521 tggtatatta tggataccac tgtatctgtg atatgtaaag tctaagttgt cgtccttgat
     2581 ggatcatgga tgagaagatg tgtcaatcac caccgccctg gatctgctag atgtgacagg
     2641 ctgccctggt tcagttatca cagtgctgat gctgtccatc ttaaaggtac agtactgtga
     2701 taactgaagg atggctgcca tcttgacatc ttacctcaga aagagatttg atttctggtg
     2761 agaaagaagt aaataacgaa gccaactgct gttactctcg cccagaaatc tgcgccaaca
     2821 tggacacacc cagtggagca agccatctga ttttcatggt cagattgtaa agaagcaatt
     2881 actcttggac agtttaagca gaaaagccac caagtcaagt cactggtcat atgatgaact
     2941 cgaagatcta ataaaagtta atttaagcgt tcattacaat aaattcaagt gaatctgttt
     3001 taagctagcc tttaacgttt ttaaattcct tcaaacaaat tagttcctct ttgcagttcc
     3061 tcatttctcg gtgagctggt gctttaatcc ctttactttt tcttcctcta gggagtgatg
     3121 gttggcatgg gacgaattaa ttcctgcagc ccgggggtac cggtcgccac catgaacacc
     3181 ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg tgcacatgga gggcaacgtg
     3241 aacggccacg ccttcgtgat cgagggcgag ggcaagggca agccctacga gggcacccag
     3301 accgccaacc tgaccgtgaa ggagggcgcc cccctgccct tcagctacga catcctgacc
     3361 accgccgtgc actacggcaa ccgggtgttc accaagtacc ccgaggacat ccccgactac
     3421 ttcaagcaga gcttccccga gggctacagc tgggagcgca ccatgacctt cgaggacaag
     3481 ggcatctgca ccatccgcag cgacatcagc ctggagggcg actgcttctt ccagaacgtg
     3541 cgcttcaagg gcaccaactt cccccccaac ggccccgtga tgcagaagaa aaccctgaag
     3601 tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc tgctggtggg caacatcaac
     3661 atggccctgc tgctggaggg cggcggccac tacctgtgcg acttcaagac cacctacaag
     3721 gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg accaccgcat cgagatcctg
     3781 ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc acgccgtggc ccgctacagc
     3841 cccctgccca gccaggtgtg gtaatctaga tcataatcag ccataccaca tttgtagagg
     3901 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3961 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4021 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4081 tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     4141 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcgg atcctgcggc
     4201 cgcaagggaa aatagaatga agtgatctcc aaaaaataag tactttttga ctgtaaataa
     4261 aattgtaagg agtaaaaagt actttttttt ctaaaaaaat gtaattaagt aaaagtaaaa
     4321 gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt
     4381 aagtacagta atcaagtaaa attactcaag tactttacac ctctggttct tgacccccta
     4441 ccttcagcaa gcccagcaga tccactagat cttttgttcc ctttagtgag ggttaattgc
     4501 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4561 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4621 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4681 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4741 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4801 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4861 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4921 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4981 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5041 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5101 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5161 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5221 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5281 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5341 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5401 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5461 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5521 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5581 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5641 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5701 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5761 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5821 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5881 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5941 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6001 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6061 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6121 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6181 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6241 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6301 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6361 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6421 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6481 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6541 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6601 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6661 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     6721 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6781 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     6841 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     6901 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6961 aaagggagcc cccgatttag agcttgacgg ggaaagccgg ccacaatgac ggctactaca
     7021 tggtgccatt ccttcctctt tataggaatg gagactacct cctgtccaac aatgctcttg
     7081 gatacgagta cgcctacctg ttggacccag gtcattgcac aacaccagaa atgccctctg
     7141 atctgcaaaa gacgtgaata tctgttcaga cacccatatc cactctgttc cacacaggtc
     7201 agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat tttactcaag
     7261 tgaaagtaca agtacttagg gaaaatttta ctcaattaaa agtaaaagta tctggctaga
     7321 atcttacttg agtaaaagta aaaaagtact ccattaaaat tgtacttgag tattaaggaa
     7381 gtaaaaggct agcaagatct gctcgagata catttgatta gaagagtttg acttgggttg
     7441 attatagcaa atagtggaac acaaacagat tttgaaatat tcttgcaaat ttatttatta
     7501 aatccattca aatattgtca ttttttgtac tgtgtaattt atattgattt gctcatacaa
     7561 tatttacatt tcgcttaaac aatacactat tatttatatt taaaaatagc tatatataaa
     7621 tcaaatgtat agctcttttt tacacaatta atcaaagcta aaatatttgt cacacaaata
     7681 cagtaagcca cttcacaatg atatcttcaa aatctgtgct tgactgaatc atttcattta
     7741 ttgttttgta attcacatta taccttacgt tctccaggca cagtttgaat tgttgataaa
     7801 tgaagataat acatctgatt taattagtct cctttgttta taataacttt aatctaattt
     7861 gatagtaata attccagcaa tacatccatt acaataaaaa tcgctttttt attttgcaca
     7921 ttagtttgcc ggtatttgtt taaagtttca ccatactttt aaatcatgtg ctatttgaaa
     7981 agggctgtta tgactggaat taaatgtttt ttttt
//
