LOCUS       sequence-182647-        7848 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 7848)
  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 7848)
  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 199-3-Dendra2
FEATURES             Location/Qualifiers
     source          1..7848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2167..2859
                     /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    2970..3091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3007..3026)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3061..3080
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3215..3402,3403..3414)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3469..3489)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3469..3487)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3508..3524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3508..3524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3521..3543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3532..3548
                     /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(3556..3562,3563..3580,3581..3586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3601..3622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3739..3756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3910..4498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3990..4009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4669..5460,5461..5529))
                     /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     5292..5311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5530..5634)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5797..5818)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6214..6225,6226..6363)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tgtattaaat aatataaaat ctttgttctt agtctgaaga ggagactgtg agagaaagct
       61 tactggttgt taaattagaa aatttgtaac ttgcagatta aaatgattta aataatttta
      121 taaaggccat gtttgaaaca aatgtaaaaa cataataaat gttaagaatt tcagatattg
      181 agattttaca atttaaaaaa agcaataaag taatagcaga gacctacact cttaaagtaa
      241 attacccaac tctgcttact agtaactttt ggaatagtta ctagtatcca ttgaacaaat
      301 actagaatct aattaaattg tgtgagtgtc caatgaaaag attcttgtat ttaaaacagt
      361 accagtatct atatatgtac aaaatttagt gctagcaaaa tgcaaataaa tactactgca
      421 tataatggat tgcattgtaa atcgactcgc cataatacat ttatttccta tttttacttt
      481 taacaaaaaa taagttcact gagtatttca tgtcattaaa tcgcattaaa cctcttgcat
      541 gctttatgta cactagtgca gttcgctaaa tgaccaccgg tgtcactaat aacacgatcg
      601 tttctgaatt ctgcattaaa cgccatttat acgttgctct tattagtaaa caatttctcc
      661 atcatgagct cgaccagtat tgcatatttc atcctcttaa atccagagtt gtttctttta
      721 atcactttct cttcatgata aaagcagttt ttggttttca gaagtttaca acaaacccag
      781 tcagttattg cctcagcaaa cactggagca actgcacact tgtgaaaagc ttcttgcgaa
      841 acacaaacct ttctctggac ttataaataa aggcattcag cagctccacc atacacctcc
      901 actggtcttg ataaagcaga tatcagcagt gatacaggta ccgccaccat ggatgaggaa
      961 atcgctgccc tggtcgttga caacggctcc ggtatgtgca aagccggttt tgctggagat
     1021 gatgcccctc gtgctgtttt cccctccatt gttggacgac ccagacatca ggtacattta
     1081 ataaaccatc ctaataacat gttaatcata ttgaaccaca ttaaactctc atttgtgcat
     1141 tttacatctt ttaaaaccga tttatattcg tttttagata atgaagttta ttaaagtgtt
     1201 aattcacagc tttattgctt gttttgagaa tctgcttgca taagtgattt ttaaacttga
     1261 tctgattttc gtttgtttcg aagttcttca gctttgaaag tcagatcttc aattcacagc
     1321 catgccatag ttttaggata cagtataagt agacgaattg ggatgcatcg tagattttat
     1381 gctgtgtttg ttatggtttt tcagtggtag agattttcag agtattatta gggttatagt
     1441 cagtgtcagg tttatttatc gtcagcgtga tgggcaaaaa acagctggca gtacgggctc
     1501 tctccacagt cgtgtgcggt cctcagggca gtgacctacg ctccagccac ctcttgcctc
     1561 cccctcgcct gcccagtgtt cagactacct gttcatcatg ctgcagctga acagtagtcc
     1621 gcacattggt taggctgggc tgggacacac acacccgcca agccgccgta agatctctgc
     1681 cattcgccgg tgaacgaata aagcaagact ttagccccgc acaatacacc gtttcagcat
     1741 cgacatcgca atgaaccaca tcgctgaatt cacaagccaa ctgctgttac tctcgcccag
     1801 aaatctgcgc caacatggac acacccagtg gagcaagcca tctgattttc atggtcagat
     1861 tgtaaagaag caattactct tggacagttt aagcagaaaa gccaccaagt caagtcactg
     1921 gtcatatgat gaactcgaag atctaataaa agttaattta agcgttcatt acaataaatt
     1981 caagtgaatc tgttttaagc tagcctttaa cgtttttaaa ttccttcaaa caaattagtt
     2041 cctctttgca gttcctcatt tctcggtgag ctggtgcttt aatcccttta ctttttcttc
     2101 ctctagggag tgatggttgg catgggacga attaattcct gcagcccggg ggtaccggtc
     2161 gccaccatga acaccccggg aattaacctg atcaaggagg acatgcgcgt gaaggtgcac
     2221 atggagggca acgtgaacgg ccacgccttc gtgatcgagg gcgagggcaa gggcaagccc
     2281 tacgagggca cccagaccgc caacctgacc gtgaaggagg gcgcccccct gcccttcagc
     2341 tacgacatcc tgaccaccgc cgtgcactac ggcaaccggg tgttcaccaa gtaccccgag
     2401 gacatccccg actacttcaa gcagagcttc cccgagggct acagctggga gcgcaccatg
     2461 accttcgagg acaagggcat ctgcaccatc cgcagcgaca tcagcctgga gggcgactgc
     2521 ttcttccaga acgtgcgctt caagggcacc aacttccccc ccaacggccc cgtgatgcag
     2581 aagaaaaccc tgaagtggga gcccagcacc gagaagctgc acgtgcgcga cggcctgctg
     2641 gtgggcaaca tcaacatggc cctgctgctg gagggcggcg gccactacct gtgcgacttc
     2701 aagaccacct acaaggccaa gaaggtggtg cagctgcccg acgcccactt cgtggaccac
     2761 cgcatcgaga tcctgggcaa cgacagcgac tacaacaagg tgaagctgta cgagcacgcc
     2821 gtggcccgct acagccccct gcccagccag gtgtggtaat ctagatcata atcagccata
     2881 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2941 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3001 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3061 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     3121 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3181 ggcggatcct gcggccgcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt
     3241 tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat
     3301 taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa
     3361 ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt
     3421 gaccccctac cttcagcaag cccagcagat ccactagatc ttttgttccc tttagtgagg
     3481 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3541 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3601 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3661 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3721 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3781 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3841 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3901 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3961 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4021 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4081 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4141 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4201 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4261 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4321 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4381 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4441 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4501 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4561 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4621 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4681 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4741 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4801 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4861 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4921 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4981 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5041 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5101 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5161 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5221 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5281 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5341 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5401 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5461 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5521 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5581 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5641 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5701 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5761 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5821 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5881 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5941 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc cacaatgacg
     6001 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
     6061 atgctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
     6121 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
     6181 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
     6241 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
     6301 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
     6361 attaaggaag taaaaggcta gcaagatctg ctcgagatac atttgattag aagagtttga
     6421 cttgggttga ttatagcaaa tagtggaaca caaacagatt ttgaaatatt cttgcaaatt
     6481 tatttattaa atccattcaa atattgtcat tttttgtact gtgtaattta tattgatttg
     6541 ctcatacaat atttacattt cgcttaaaca atacactatt atttatattt aaaaatagct
     6601 atatataaat caaatgtata gctctttttt acacaattaa tcaaagctaa aatatttgtc
     6661 acacaaatac agtaagccac ttcacaatga tatcttcaaa atctgtgctt gactgaatca
     6721 tttcatttat tgttttgtaa ttcacattat accttacgtt ctccaggcac agtttgaatt
     6781 gttgataaat gaagataata catctgattt aattagtctc ctttgtttat aataacttta
     6841 atctaatttg atagtaataa ttccagcaat acatccatta caataaaaat cgctttttta
     6901 ttttgcacat tagtttgccg gtatttgttt aaagtttcac catactttta aatcatgtgc
     6961 tatttgaaaa gggctgttat gactggaatt aaatgttttt ttttatatca gtgttttaga
     7021 ttgaagtaca atatttatag gtaaaattca ccagcatatg cttaaagata gccatgctcc
     7081 aggtccttgt gtccttctaa ggtctgtttg tggtttaagc tgtttgttga cacacgcttg
     7141 tcatctccat ttttctgtga agttattgtc agtttattgc acactgggtt gcattaagct
     7201 gcagtttttt cacaacagtt ttccacattg taacagtcag tctccaaatt attccatcag
     7261 tcctcaaatt tagtcatttg aggccatgaa aacacttaaa actacatgaa ggtagtttgg
     7321 cattaggttt tagacttgtt cttgtttggc atattgcaac ctgtgtaaaa atgatttgat
     7381 ttttattttt tttttttttt tttcagaaag aaacggtcca aaaagagtta tgtgaacatt
     7441 tatccatgtg tttaatgttt tttaaattaa aaaaaaacat ttttattaaa ccacttgtga
     7501 ctaaaccttt tttagttatt ctattttgtg acgagtctga atggccaaag tactcttgaa
     7561 tccaaaatct ttatagtaaa atgtttaatc ttacttgcag tgttgggtat aatttgtaac
     7621 aaagttgaat tccatatctg ctgagtgaaa aagtaaagta agagattgct gtaaatattt
     7681 aggttattta tttattaata caatatagaa agtaacaaaa cacaatgtaa aaaatcaata
     7741 aagaaagtaa atattaaaag taatgataaa ctaatgatgt aaacaatatt agcaacaaag
     7801 tttgtacaat tcagggaggg taaagaattt cacctaatag tcaaactc
//
