LOCUS       sequence-182574-        8183 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 8183)
  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 8183)
  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 19c-Dendra2
FEATURES             Location/Qualifiers
     source          1..8183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1738..1747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3297..3989
                     /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    4100..4221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4137..4156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4191..4210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4345..4532,4533..4544)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4599..4619)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4599..4617)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4638..4654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4638..4654)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4651..4673)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4662..4678
                     /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(4686..4692,4693..4710,4711..4716))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4731..4752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4869..4886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5040..5628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5120..5139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5799..6590,6591..6659))
                     /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     6422..6441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6660..6764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6927..6948)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7344..7355,7356..7493)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gcatatgctt aaagatagcc atgctccagg tccttgtgtc cttctaaggt ctgtttgtgg
       61 tttaagctgt ttgttgacac acgcttgtca tctccatttt tctgtgaagt tattgtcagt
      121 ttattgcaca ctgggttgca ttaagctgca gttttttcac aacagttttc cacattgtaa
      181 cagtcagtct ccaaattatt ccatcagtcc tcaaatttag tcatttgagg ccatgaaaac
      241 acttaaaact acatgaaggt agtttggcat taggttttag acttgttctt gtttggcata
      301 ttgcaacctg tgtaaaaatg atttgatttt tatttttttt tttttttttt cagaaagaaa
      361 cggtccaaaa agagttatgt gaacatttat ccatgtgttt aatgtttttt aaattaaaaa
      421 aaaacatttt tattaaacca cttgtgacta aacctttttt agttattcta ttttgtgacg
      481 agtctgaatg gccaaagtac tcttgaatcc aaaatcttta tagtaaaatg tttaatctta
      541 cttgcagtgt tgggtataat ttgtaacaaa gttgaattcc atatctgctg agtgaaaaag
      601 taaagtaaga gattgctgta aatatttagg ttatttattt attaatacaa tatagaaagt
      661 aacaaaacac aatgtaaaaa atcaataaag aaagtaaata ttaaaagtaa tgataaacta
      721 atgatgtaaa caatattagc aacaaagttt gtacaattca gggagggtaa agaatttcac
      781 ctaatagtca aactctgtat taaataatat aaaatctttg ttcttagtct gaagaggaga
      841 ctgtgagaga aagcttactg gttgttaaat tagaaaattt gtaacttgca gattaaaatg
      901 atttaaataa ttttataaag gccatgtttg aaacaaatgt aaaaacataa taaatgttaa
      961 gaatttcaga tattgagatt ttacaattta aaaaaagcaa taaagtaata gcagagacct
     1021 acactcttaa agtaaattac ccaactctgc ttactagtaa cttttggaat agttactagt
     1081 atccattgaa caaatactag aatctaatta aattgtgtga gtgtccaatg aaaagattct
     1141 tgtatttaaa acagtaccag tatctatata tgtacaaaat ttagtgctag caaaatgcaa
     1201 ataaatacta ctgcatataa tggattgcat tgtaaatcga ctcgccataa tacatttatt
     1261 tcctattttt acttttaaca aaaaataagt tcactgagta tttcatgtca ttaaatcgca
     1321 ttaaacctct tgcatgcttt atgtacacta gtgcagttcg ctaaatgacc accggtgtca
     1381 ctaataacac gatcgtttct gaattctgca ttaaacgcca tttatacgtt gctcttatta
     1441 gtaaacaatt tctccatcat gagctcgacc agtattgcat atttcatcct cttaaatcca
     1501 gagttgtttc ttttaatcac tttctcttca tgataaaagc agtttttggt tttcagaagt
     1561 ttacaacaaa cccagtcagt tattgcctca gcaaacactg gagcaactgc acacttgtga
     1621 aaagcttctt gcgaaacaca aacctttctc tggacttata aataaaggca ttcagcagct
     1681 ccaccataca cctccactgg tcttgataaa gcagatatca gcagtgatac aggtaccgcc
     1741 accatggatg aggaaatcgc tgccctggtc gttgacaacg gctccggtat gtgcaaagcc
     1801 ggttttgctg gagatgatgc ccctcgtgct gttttcccct ccattgttgg acgacccaga
     1861 catcaggtac atttaataaa ccatcctaat aacatgttaa tcatattgaa ccacattaaa
     1921 ctctcatttg tgcattttac atcttttaaa accgatttat attcgttttt agataatgaa
     1981 gtttattaaa gtgttaattc acagctttat tgcttgtttt gagaatctgc ttgcataagt
     2041 gatttttaaa cttgatctga ttttcgtttg tttcgaagtt cttcagcttt gaaagtcaga
     2101 tcttcaattc acagccatgc catagtttta ggatacagta taagtagacg aattgggatg
     2161 catcgtagat tttatgctgt gtttgttatg gtttttcagt ggtagagatt ttcagagtat
     2221 tattagggtt atagtcagtg tcaggtttat ttatcgtcag cgtgatgggc aaaaaacagc
     2281 tggcagtacg ggctcatcca cccgaaaaca gcaagacaaa tatttgcatt taaatatatt
     2341 tttaaaatca tgcaaaaaca ttttaaatat atttttgaga agtagcatta ttacaagcaa
     2401 ttgtcactta ggactactac acagaaaact tagcatctta ttttcttact atactagtgt
     2461 catagaatta tggcatatca tacagacttt atttccaaaa gagttttgca taaacttttt
     2521 gcattcacat gactagaaat ataatcttga ttgtagaagt ggtttgtccc tggcgcagcg
     2581 agttttgcat ggatttgcac agcatgttgt aataaagccg gatgcaatcc tgcaaaactc
     2641 accccagata acagggcttt tccataagca gcatctagtg cagctccaca gtggcaggca
     2701 gaaggcgtcc ggcaatactt gaagtgcaga cattgcagta gatccactgg cactacctgc
     2761 actgtgagca ctttggaact gccaggacaa actcaacaca gatcataaaa gaaagaataa
     2821 ttaaaacaaa acaataataa ttgatcaaac aagaactgca tgttatagaa cttgatgata
     2881 gtatgtttgc ttccgtgtgc tgaaagccaa ctgctgttac tctcgcccag aaatctgcgc
     2941 caacatggac acacccagtg gagcaagcca tctgattttc atggtcagat tgtaaagaag
     3001 caattactct tggacagttt aagcagaaaa gccaccaagt caagtcactg gtcatatgat
     3061 gaactcgaag atctaataaa agttaattta agcgttcatt acaataaatt caagtgaatc
     3121 tgttttaagc tagcctttaa cgtttttaaa ttccttcaaa caaattagtt cctctttgca
     3181 gttcctcatt tctcggtgag ctggtgcttt aatcccttta ctttttcttc ctctagggag
     3241 tgatggttgg catgggacga attaattcct gcagcccggg ggtaccggtc gccaccatga
     3301 acaccccggg aattaacctg atcaaggagg acatgcgcgt gaaggtgcac atggagggca
     3361 acgtgaacgg ccacgccttc gtgatcgagg gcgagggcaa gggcaagccc tacgagggca
     3421 cccagaccgc caacctgacc gtgaaggagg gcgcccccct gcccttcagc tacgacatcc
     3481 tgaccaccgc cgtgcactac ggcaaccggg tgttcaccaa gtaccccgag gacatccccg
     3541 actacttcaa gcagagcttc cccgagggct acagctggga gcgcaccatg accttcgagg
     3601 acaagggcat ctgcaccatc cgcagcgaca tcagcctgga gggcgactgc ttcttccaga
     3661 acgtgcgctt caagggcacc aacttccccc ccaacggccc cgtgatgcag aagaaaaccc
     3721 tgaagtggga gcccagcacc gagaagctgc acgtgcgcga cggcctgctg gtgggcaaca
     3781 tcaacatggc cctgctgctg gagggcggcg gccactacct gtgcgacttc aagaccacct
     3841 acaaggccaa gaaggtggtg cagctgcccg acgcccactt cgtggaccac cgcatcgaga
     3901 tcctgggcaa cgacagcgac tacaacaagg tgaagctgta cgagcacgcc gtggcccgct
     3961 acagccccct gcccagccag gtgtggtaat ctagatcata atcagccata ccacatttgt
     4021 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     4081 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4141 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4201 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     4261 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcggatcct
     4321 gcggccgcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta
     4381 aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag
     4441 taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta
     4501 agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac
     4561 cttcagcaag cccagcagat ccactagatc ttttgttccc tttagtgagg gttaattgcg
     4621 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4681 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4741 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4801 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4861 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4921 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4981 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5041 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5101 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5161 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5221 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5281 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5341 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5401 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5461 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5521 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5581 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5641 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5701 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5761 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5821 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5881 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5941 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6001 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6061 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6541 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6601 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6661 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6721 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6781 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6841 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6901 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6961 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     7021 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     7081 aagggagccc ccgatttaga gcttgacggg gaaagccggc cacaatgacg gctactacat
     7141 ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca atgctcttgg
     7201 atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga
     7261 tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca
     7321 gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt
     7381 gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa
     7441 tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag
     7501 taaaaggcta gcaagatctg ctcgagatac atttgattag aagagtttga cttgggttga
     7561 ttatagcaaa tagtggaaca caaacagatt ttgaaatatt cttgcaaatt tatttattaa
     7621 atccattcaa atattgtcat tttttgtact gtgtaattta tattgatttg ctcatacaat
     7681 atttacattt cgcttaaaca atacactatt atttatattt aaaaatagct atatataaat
     7741 caaatgtata gctctttttt acacaattaa tcaaagctaa aatatttgtc acacaaatac
     7801 agtaagccac ttcacaatga tatcttcaaa atctgtgctt gactgaatca tttcatttat
     7861 tgttttgtaa ttcacattat accttacgtt ctccaggcac agtttgaatt gttgataaat
     7921 gaagataata catctgattt aattagtctc ctttgtttat aataacttta atctaatttg
     7981 atagtaataa ttccagcaat acatccatta caataaaaat cgctttttta ttttgcacat
     8041 tagtttgccg gtatttgttt aaagtttcac catactttta aatcatgtgc tatttgaaaa
     8101 gggctgttat gactggaatt aaatgttttt ttttatatca gtgttttaga ttgaagtaca
     8161 atatttatag gtaaaattca cca
//
