LOCUS       sequence-182661-        7973 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 7973)
  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 7973)
  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 212-Dendra2
FEATURES             Location/Qualifiers
     source          1..7973
                     /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             3047..3739
                     /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    3850..3971
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3887..3906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3941..3960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4095..4282,4283..4294)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4349..4369)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4349..4367)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4388..4404)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4388..4404)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4401..4423)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4412..4428
                     /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(4436..4442,4443..4460,4461..4466))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4481..4502
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4619..4636)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4790..5378)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4870..4889)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5549..6340,6341..6409))
                     /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     6172..6191
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6410..6514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6677..6698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7094..7105,7106..7243)
                     /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 ggctttttca aggctcagac aaaccattta
     2281 cacggaaaaa aactgaatgc ttgaaaagtc agtgcatcaa taccttggct ctagactgct
     2341 tactgctaaa tgtgtctgaa agtacagtaa cagtctacag tcatggctac tgacgtctgg
     2401 caaccttcca cagaggattc aagtgttcag agaacatgaa gaatctgtga agtccaggtc
     2461 agttcagctt taagagcaga aatgtgtgtc atccactctg aacatgtggg ttgcggtgca
     2521 gccggtcagc caatcagata cgcccgtcta ctgtttccac tgccaaccct cctgctgtct
     2581 ccatggcgac cgtggcatta gattgttact gtaggaacag aatttttggt aacagtctac
     2641 agccatggtc gctaagccaa ctgctgttac tctcgcccag aaatctgcgc caacatggac
     2701 acacccagtg gagcaagcca tctgattttc atggtcagat tgtaaagaag caattactct
     2761 tggacagttt aagcagaaaa gccaccaagt caagtcactg gtcatatgat gaactcgaag
     2821 atctaataaa agttaattta agcgttcatt acaataaatt caagtgaatc tgttttaagc
     2881 tagcctttaa cgtttttaaa ttccttcaaa caaattagtt cctctttgca gttcctcatt
     2941 tctcggtgag ctggtgcttt aatcccttta ctttttcttc ctctagggag tgatggttgg
     3001 catgggacga attaattcct gcagcccggg ggtaccggtc gccaccatga acaccccggg
     3061 aattaacctg atcaaggagg acatgcgcgt gaaggtgcac atggagggca acgtgaacgg
     3121 ccacgccttc gtgatcgagg gcgagggcaa gggcaagccc tacgagggca cccagaccgc
     3181 caacctgacc gtgaaggagg gcgcccccct gcccttcagc tacgacatcc tgaccaccgc
     3241 cgtgcactac ggcaaccggg tgttcaccaa gtaccccgag gacatccccg actacttcaa
     3301 gcagagcttc cccgagggct acagctggga gcgcaccatg accttcgagg acaagggcat
     3361 ctgcaccatc cgcagcgaca tcagcctgga gggcgactgc ttcttccaga acgtgcgctt
     3421 caagggcacc aacttccccc ccaacggccc cgtgatgcag aagaaaaccc tgaagtggga
     3481 gcccagcacc gagaagctgc acgtgcgcga cggcctgctg gtgggcaaca tcaacatggc
     3541 cctgctgctg gagggcggcg gccactacct gtgcgacttc aagaccacct acaaggccaa
     3601 gaaggtggtg cagctgcccg acgcccactt cgtggaccac cgcatcgaga tcctgggcaa
     3661 cgacagcgac tacaacaagg tgaagctgta cgagcacgcc gtggcccgct acagccccct
     3721 gcccagccag gtgtggtaat ctagatcata atcagccata ccacatttgt agaggtttta
     3781 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3841 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3901 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3961 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4021 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcggatcct gcggccgcaa
     4081 gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg
     4141 taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt
     4201 gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa
     4261 tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac cttcagcaag
     4321 cccagcagat ccactagatc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     4381 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4441 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4501 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4561 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4621 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4681 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4741 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4801 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4861 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4921 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4981 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5041 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5101 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5161 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5221 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5281 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5341 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5401 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5461 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5521 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5581 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5641 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5701 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5761 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5821 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5881 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5941 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6001 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6061 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6121 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6181 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6241 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6301 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6361 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6421 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6481 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     6541 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     6601 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     6661 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     6721 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     6781 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     6841 ccgatttaga gcttgacggg gaaagccggc cacaatgacg gctactacat ggtgccattc
     6901 cttcctcttt ataggaatgg agactacctc ctgtccaaca atgctcttgg atacgagtac
     6961 gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga tctgcaaaag
     7021 acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca gaggtttgtc
     7081 caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa
     7141 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga
     7201 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaaggcta
     7261 gcaagatctg ctcgagatac atttgattag aagagtttga cttgggttga ttatagcaaa
     7321 tagtggaaca caaacagatt ttgaaatatt cttgcaaatt tatttattaa atccattcaa
     7381 atattgtcat tttttgtact gtgtaattta tattgatttg ctcatacaat atttacattt
     7441 cgcttaaaca atacactatt atttatattt aaaaatagct atatataaat caaatgtata
     7501 gctctttttt acacaattaa tcaaagctaa aatatttgtc acacaaatac agtaagccac
     7561 ttcacaatga tatcttcaaa atctgtgctt gactgaatca tttcatttat tgttttgtaa
     7621 ttcacattat accttacgtt ctccaggcac agtttgaatt gttgataaat gaagataata
     7681 catctgattt aattagtctc ctttgtttat aataacttta atctaatttg atagtaataa
     7741 ttccagcaat acatccatta caataaaaat cgctttttta ttttgcacat tagtttgccg
     7801 gtatttgttt aaagtttcac catactttta aatcatgtgc tatttgaaaa gggctgttat
     7861 gactggaatt aaatgttttt ttttatatca gtgttttaga ttgaagtaca atatttatag
     7921 gtaaaattca ccagcatatg cttaaagata gccatgctcc aggtccttgt gtc
//
