LOCUS       sequence-182771-        8049 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 8049)
  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 8049)
  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 130c-1-Dendra2
FEATURES             Location/Qualifiers
     source          1..8049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1764..1773
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3188..3880
                     /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    3991..4112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4028..4047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4082..4101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4236..4423,4424..4435)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4490..4510)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4490..4508)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4529..4545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4529..4545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4542..4564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4553..4569
                     /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(4577..4583,4584..4601,4602..4607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4622..4643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4760..4777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4931..5519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5011..5030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5690..6481,6482..6550))
                     /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     6313..6332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6551..6655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6818..6839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7235..7246,7247..7384)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 caatatttat aggtaaaatt caccagcata tgcttaaaga tagccatgct ccaggtcctt
       61 gtgtccttct aaggtctgtt tgtggtttaa gctgtttgtt gacacacgct tgtcatctcc
      121 atttttctgt gaagttattg tcagtttatt gcacactggg ttgcattaag ctgcagtttt
      181 ttcacaacag ttttccacat tgtaacagtc agtctccaaa ttattccatc agtcctcaaa
      241 tttagtcatt tgaggccatg aaaacactta aaactacatg aaggtagttt ggcattaggt
      301 tttagacttg ttcttgtttg gcatattgca acctgtgtaa aaatgatttg atttttattt
      361 tttttttttt tttttncaga aagaaacggt ccaaaaagag ttatgtgaac atttatccat
      421 gtgtttaatg ttttttaaat taaaaaaaaa catttttatt aaaccacttg tgactaaacc
      481 ttttttagtt attctatttt gtgacgagtc tgaatggcca aagtactctt gaatccaaaa
      541 tctttatagt aaaatgttta atcttacttg cagtgttggg tataatttgt aacaaagttg
      601 aattccatat ctgctgagtg aaaaagtaaa gtaagagatt gctgtaaata tttaggttat
      661 ttatttatta atacaatata gaaagtaaca aaacacaatg taaaaaatca ataaagaaag
      721 taaatattaa aagtaatgat aaactaatga tgtaaacaat attagcaaca aagtttgtac
      781 aattcaggga gggtaaagaa tttcacctaa tagtcaaact ctgtattaaa taatataaaa
      841 tctttgttct tagtctgaag aggagactgt gagagaaagc ttactggttg ttaaattaga
      901 aaatttgtaa cttgcagatt aaaatgattt aaataatttt ataaaggcca tgtttgaaac
      961 aaatgtaaaa acataataaa tgttaagaat ttcagatatt gagattttac aatttaaaaa
     1021 aagcaataaa gtaatagcag agacctacac tcttaaagta aattacccaa ctctgcttac
     1081 tagtaacttt tggaatagtt actagtatcc attgaacaaa tactagaatc taattaaatt
     1141 gtgtgagtgt ccaatgaaaa gattcttgta tttaaaacag taccagtatc tatatatgta
     1201 caaaatttag tgctagcaaa atgcaaataa atactactgc atataatgga ttgcattgta
     1261 aatcgactcg ccataataca tttatttcct atttttactt ttaacaaaaa ataagttcac
     1321 tgagtatttc atgtcattaa atcgcattaa acctcttgca tgctttatgt acactagtgc
     1381 agttcgctaa atgaccaccg gtgtcactaa taacacgatc gtttctgaat tctgcattaa
     1441 acgccattta tacgttgctc ttattagtaa acaatttctc catcatgagc tcgaccagta
     1501 ttgcatattt catcctctta aatccagagt tgtttctttt aatcactttc tcttcatgat
     1561 aaaagcagtt tttggttttc agaagtttac aacaaaccca gtcagttatt gcctcagcaa
     1621 acactggagc aactgcacac ttgtgaaaag cttcttgcga aacacaaacc tttctctgga
     1681 cttataaata aaggcattca gcagctccac catacacctc cactggtctt gataaagcag
     1741 atatcagcag tgatacaggt accgccacca tggatgagga aatcgctgcc ctggtcgttg
     1801 acaacggctc cggtatgtgc aaagccggtt ttgctggaga tgatgcccct cgtgctgttt
     1861 tcccctccat tgttggacga cccagacatc aggtacattt aataaaccat cctaataaca
     1921 tgttaatcat attgaaccac attaaactct catttgtgca ttttacatct tttaaaaccg
     1981 atttatattc gtttttagat aatgaagttt attaaagtgt taattcacag ctttattgct
     2041 tgttttgaga atctgcttgc ataagtgatt tttaaacttg atctgatttt cgtttgtttc
     2101 gaagttcttc agctttgaaa gtcagatctt caattcacag ccatgccata gttttaggat
     2161 acagtataag tagacgaatt gggatgcatc gtagatttta tgctgtgttt gttatggttt
     2221 ttcagtggta gagattttca gagtattatt agggttatag tcagtgtcag gtttatttat
     2281 cgtcagcgtg atgggcaaaa aacagctggc agtacgggct ttgtaaagca gagcccctca
     2341 ggatccataa atgaagtgaa aactttgctt ttcctcacca tttgctttct caaaccttct
     2401 ggttaaatct gttaatctgc tattgcagtc atttagttag agttcaatgc attaatcatt
     2461 ttgtttttga agtcatgatt taactgatta acgtaagctg ctcttttcct tgatttttca
     2521 accaaatgtg cataataatc aattaaagac tgtagaaatc aaatgcaatg gtaaagcatg
     2581 atgagggaga ggtctgtacg gtgatattga ccatgttttg tccattgccc tttttctgtt
     2641 gtactactgg ccaatcagaa gagcagtgca atattaaaag ggcattggct gatagaacag
     2701 agtccttcac ctctcaccct ctcctgctct gacattattt aacatgtgca gcgacccacg
     2761 cctgcaaagc tggacaaccg tcaaagtcac tcgctagcca actgctgtta ctctcgccca
     2821 gaaatctgcg ccaacatgga cacacccagt ggagcaagcc atctgatttt catggtcaga
     2881 ttgtaaagaa gcaattactc ttggacagtt taagcagaaa agccaccaag tcaagtcact
     2941 ggtcatatga tgaactcgaa gatctaataa aagttaattt aagcgttcat tacaataaat
     3001 tcaagtgaat ctgttttaag ctagccttta acgtttttaa attccttcaa acaaattagt
     3061 tcctctttgc agttcctcat ttctcggtga gctggtgctt taatcccttt actttttctt
     3121 cctctaggga gtgatggttg gcatgggacg aattaattcc tgcagcccgg gggtaccggt
     3181 cgccaccatg aacaccccgg gaattaacct gatcaaggag gacatgcgcg tgaaggtgca
     3241 catggagggc aacgtgaacg gccacgcctt cgtgatcgag ggcgagggca agggcaagcc
     3301 ctacgagggc acccagaccg ccaacctgac cgtgaaggag ggcgcccccc tgcccttcag
     3361 ctacgacatc ctgaccaccg ccgtgcacta cggcaaccgg gtgttcacca agtaccccga
     3421 ggacatcccc gactacttca agcagagctt ccccgagggc tacagctggg agcgcaccat
     3481 gaccttcgag gacaagggca tctgcaccat ccgcagcgac atcagcctgg agggcgactg
     3541 cttcttccag aacgtgcgct tcaagggcac caacttcccc cccaacggcc ccgtgatgca
     3601 gaagaaaacc ctgaagtggg agcccagcac cgagaagctg cacgtgcgcg acggcctgct
     3661 ggtgggcaac atcaacatgg ccctgctgct ggagggcggc ggccactacc tgtgcgactt
     3721 caagaccacc tacaaggcca agaaggtggt gcagctgccc gacgcccact tcgtggacca
     3781 ccgcatcgag atcctgggca acgacagcga ctacaacaag gtgaagctgt acgagcacgc
     3841 cgtggcccgc tacagccccc tgcccagcca ggtgtggtaa tctagatcat aatcagccat
     3901 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     3961 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4021 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4081 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     4141 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4201 cggcggatcc tgcggccgca agggaaaata gaatgaagtg atctccaaaa aataagtact
     4261 ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa
     4321 ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa
     4381 aataatactt aagtacagta atcaagtaaa attactcaag tactttacac ctctggttct
     4441 tgacccccta ccttcagcaa gcccagcaga tccactagat cttttgttcc ctttagtgag
     4501 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4561 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4621 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4681 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4741 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4801 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4861 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4921 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5101 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5341 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5521 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5581 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5641 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5701 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5761 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5821 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5881 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5941 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6001 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6061 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6121 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6181 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6241 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6301 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6361 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6421 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6481 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6541 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6601 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6661 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     6721 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     6781 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     6841 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6901 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6961 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg ccacaatgac
     7021 ggctactaca tggtgccatt ccttcctctt tataggaatg gagactacct cctgtccaac
     7081 aatgctcttg gatacgagta cgcctacctg ttggacccag gtcattgcac aacaccagaa
     7141 atgccctctg atctgcaaaa gacgtgaata tctgttcaga cacccatatc cactctgttc
     7201 cacacaggtc agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat
     7261 tttactcaag tgaaagtaca agtacttagg gaaaatttta ctcaattaaa agtaaaagta
     7321 tctggctaga atcttacttg agtaaaagta aaaaagtact ccattaaaat tgtacttgag
     7381 tattaaggaa gtaaaaggct agcaagatct gctcgagata catttgatta gaagagtttg
     7441 acttgggttg attatagcaa atagtggaac acaaacagat tttgaaatat tcttgcaaat
     7501 ttatttatta aatccattca aatattgtca ttttttgtac tgtgtaattt atattgattt
     7561 gctcatacaa tatttacatt tcgcttaaac aatacactat tatttatatt taaaaatagc
     7621 tatatataaa tcaaatgtat agctcttttt tacacaatta atcaaagcta aaatatttgt
     7681 cacacaaata cagtaagcca cttcacaatg atatcttcaa aatctgtgct tgactgaatc
     7741 atttcattta ttgttttgta attcacatta taccttacgt tctccaggca cagtttgaat
     7801 tgttgataaa tgaagataat acatctgatt taattagtct cctttgttta taataacttt
     7861 aatctaattt gatagtaata attccagcaa tacatccatt acaataaaaa tcgctttttt
     7921 attttgcaca ttagtttgcc ggtatttgtt taaagtttca ccatactttt aaatcatgtg
     7981 ctatttgaaa agggctgtta tgactggaat taaatgtttt tttttatatc agtgttttag
     8041 attgaagta
//
