LOCUS       sequence-182639-        8141 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 8141)
  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 8141)
  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 146a-Dendra2
FEATURES             Location/Qualifiers
     source          1..8141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1631..1640
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3147..3839
                     /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    3950..4071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3987..4006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4041..4060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4195..4382,4383..4394)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4449..4469)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4449..4467)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4488..4504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4488..4504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4501..4523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4512..4528
                     /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(4536..4542,4543..4560,4561..4566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4581..4602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4719..4736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4890..5478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4970..4989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5649..6440,6441..6509))
                     /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     6272..6291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6510..6614)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6777..6798)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7194..7205,7206..7343)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gttattgtca gtttattgca cactgggttg cattaagctg cagttttttc acaacagttt
       61 tccacattgt aacagtcagt ctccaaatta ttccatcagt cctcaaattt agtcatttga
      121 ggccatgaaa acacttaaaa ctacatgaag gtagtttggc attaggtttt agacttgttc
      181 ttgtttggca tattgcaacc tgtgtaaaaa tgatttgatt tttatttttt tttttttttt
      241 tttcagaaag aaacggtcca aaaagagtta tgtgaacatt tatccatgtg tttaatgttt
      301 tttaaattaa aaaaaaacat ttttattaaa ccacttgtga ctaaaccttt tttagttatt
      361 ctattttgtg acgagtctga atggccaaag tactcttgaa tccaaaatct ttatagtaaa
      421 atgtttaatc ttacttgcag tgttgggtat aatttgtaac aaagttgaat tccatatctg
      481 ctgagtgaaa aagtaaagta agagattgct gtaaatattt aggttattta tttattaata
      541 caatatagaa agtaacaaaa cacaatgtaa aaaatcaata aagaaagtaa atattaaaag
      601 taatgataaa ctaatgatgt aaacaatatt agcaacaaag tttgtacaat tcagggaggg
      661 taaagaattt cacctaatag tcaaactctg tattaaataa tataaaatct ttgttcttag
      721 tctgaagagg agactgtgag agaaagctta ctggttgtta aattagaaaa tttgtaactt
      781 gcagattaaa atgatttaaa taattttata aaggccatgt ttgaaacaaa tgtaaaaaca
      841 taataaatgt taagaatttc agatattgag attttacaat ttaaaaaaag caataaagta
      901 atagcagaga cctacactct taaagtaaat tacccaactc tgcttactag taacttttgg
      961 aatagttact agtatccatt gaacaaatac tagaatctaa ttaaattgtg tgagtgtcca
     1021 atgaaaagat tcttgtattt aaaacagtac cagtatctat atatgtacaa aatttagtgc
     1081 tagcaaaatg caaataaata ctactgcata taatggattg cattgtaaat cgactcgcca
     1141 taatacattt atttcctatt tttactttta acaaaaaata agttcactga gtatttcatg
     1201 tcattaaatc gcattaaacc tcttgcatgc tttatgtaca ctagtgcagt tcgctaaatg
     1261 accaccggtg tcactaataa cacgatcgtt tctgaattct gcattaaacg ccatttatac
     1321 gttgctctta ttagtaaaca atttctccat catgagctcg accagtattg catatttcat
     1381 cctcttaaat ccagagttgt ttcttttaat cactttctct tcatgataaa agcagttttt
     1441 ggttttcaga agtttacaac aaacccagtc agttattgcc tcagcaaaca ctggagcaac
     1501 tgcacacttg tgaaaagctt cttgcgaaac acaaaccttt ctctggactt ataaataaag
     1561 gcattcagca gctccaccat acacctccac tggtcttgat aaagcagata tcagcagtga
     1621 tacaggtacc gccaccatgg atgaggaaat cgctgccctg gtcgttgaca acggctccgg
     1681 tatgtgcaaa gccggttttg ctggagatga tgcccctcgt gctgttttcc cctccattgt
     1741 tggacgaccc agacatcagg tacatttaat aaaccatcct aataacatgt taatcatatt
     1801 gaaccacatt aaactctcat ttgtgcattt tacatctttt aaaaccgatt tatattcgtt
     1861 tttagataat gaagtttatt aaagtgttaa ttcacagctt tattgcttgt tttgagaatc
     1921 tgcttgcata agtgattttt aaacttgatc tgattttcgt ttgtttcgaa gttcttcagc
     1981 tttgaaagtc agatcttcaa ttcacagcca tgccatagtt ttaggataca gtataagtag
     2041 acgaattggg atgcatcgta gattttatgc tgtgtttgtt atggtttttc agtggtagag
     2101 attttcagag tattattagg gttatagtca gtgtcaggtt tatttatcgt cagcgtgatg
     2161 ggcaaaaaac agctggcagt acgggctccc ttgcggagaa acacctatga atttataatc
     2221 agcgtttctc atgaccgagg aaaacagcag cagcagataa gtgtgtctca aatcatcagg
     2281 gcttcttgtt tgtctgttag tgtattaaac atgtataata atttgaagtt tggagaagtt
     2341 ttcaggtttt attatgaatt tttttaaacg cactgaacaa tagcctactg tttcattatt
     2401 aattaaagtt tcattaatgg tggctgagtt tgttttgagc acttttccct gagaactgaa
     2461 ttccatagat ggtgttcatg aaaagttcat ctatgggctc agttcttctg gcaatctgtt
     2521 taatgtctgc tacaaattct gaaaaaactg gcatacatac catttttatt ctgttatatt
     2581 tgtgtgtgtg tgtgtgtgtg tgttatatat tattgtgttt ttatttagct gttcttgtac
     2641 tgtttgtttg tttgttttgt gtattcctgt attttaatta ttctaaaaat tttgaaagtc
     2701 gaaatgtaaa agtataaaaa tgaaaacaat gtgatattgg agaccacttt aaagagccaa
     2761 ctgctgttac tctcgcccag aaatctgcgc caacatggac acacccagtg gagcaagcca
     2821 tctgattttc atggtcagat tgtaaagaag caattactct tggacagttt aagcagaaaa
     2881 gccaccaagt caagtcactg gtcatatgat gaactcgaag atctaataaa agttaattta
     2941 agcgttcatt acaataaatt caagtgaatc tgttttaagc tagcctttaa cgtttttaaa
     3001 ttccttcaaa caaattagtt cctctttgca gttcctcatt tctcggtgag ctggtgcttt
     3061 aatcccttta ctttttcttc ctctagggag tgatggttgg catgggacga attaattcct
     3121 gcagcccggg ggtaccggtc gccaccatga acaccccggg aattaacctg atcaaggagg
     3181 acatgcgcgt gaaggtgcac atggagggca acgtgaacgg ccacgccttc gtgatcgagg
     3241 gcgagggcaa gggcaagccc tacgagggca cccagaccgc caacctgacc gtgaaggagg
     3301 gcgcccccct gcccttcagc tacgacatcc tgaccaccgc cgtgcactac ggcaaccggg
     3361 tgttcaccaa gtaccccgag gacatccccg actacttcaa gcagagcttc cccgagggct
     3421 acagctggga gcgcaccatg accttcgagg acaagggcat ctgcaccatc cgcagcgaca
     3481 tcagcctgga gggcgactgc ttcttccaga acgtgcgctt caagggcacc aacttccccc
     3541 ccaacggccc cgtgatgcag aagaaaaccc tgaagtggga gcccagcacc gagaagctgc
     3601 acgtgcgcga cggcctgctg gtgggcaaca tcaacatggc cctgctgctg gagggcggcg
     3661 gccactacct gtgcgacttc aagaccacct acaaggccaa gaaggtggtg cagctgcccg
     3721 acgcccactt cgtggaccac cgcatcgaga tcctgggcaa cgacagcgac tacaacaagg
     3781 tgaagctgta cgagcacgcc gtggcccgct acagccccct gcccagccag gtgtggtaat
     3841 ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3901 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4021 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     4081 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4141 ttaaccaata ggccgaaatc ggcggatcct gcggccgcaa gggaaaatag aatgaagtga
     4201 tctccaaaaa ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt
     4261 tttttctaaa aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag
     4321 taaagtaaaa atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt
     4381 actttacacc tctggttctt gaccccctac cttcagcaag cccagcagat ccactagatc
     4441 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     4501 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4561 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4621 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4681 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5521 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5581 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5641 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5701 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5761 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5821 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5881 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5941 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6001 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6061 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6121 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6181 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6241 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6301 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6361 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6421 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6481 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6541 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6601 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     6661 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     6721 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     6781 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     6841 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     6901 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     6961 gaaagccggc cacaatgacg gctactacat ggtgccattc cttcctcttt ataggaatgg
     7021 agactacctc ctgtccaaca atgctcttgg atacgagtac gcctacctgt tggacccagg
     7081 tcattgcaca acaccagaaa tgccctctga tctgcaaaag acgtgaatat ctgttcagac
     7141 acccatatcc actctgttcc acacaggtca gaggtttgtc caggagttct tgacagaggt
     7201 gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac
     7261 tcaattaaaa gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc
     7321 cattaaaatt gtacttgagt attaaggaag taaaaggcta gcaagatctg ctcgagatac
     7381 atttgattag aagagtttga cttgggttga ttatagcaaa tagtggaaca caaacagatt
     7441 ttgaaatatt cttgcaaatt tatttattaa atccattcaa atattgtcat tttttgtact
     7501 gtgtaattta tattgatttg ctcatacaat atttacattt cgcttaaaca atacactatt
     7561 atttatattt aaaaatagct atatataaat caaatgtata gctctttttt acacaattaa
     7621 tcaaagctaa aatatttgtc acacaaatac agtaagccac ttcacaatga tatcttcaaa
     7681 atctgtgctt gactgaatca tttcatttat tgttttgtaa ttcacattat accttacgtt
     7741 ctccaggcac agtttgaatt gttgataaat gaagataata catctgattt aattagtctc
     7801 ctttgtttat aataacttta atctaatttg atagtaataa ttccagcaat acatccatta
     7861 caataaaaat cgctttttta ttttgcacat tagtttgccg gtatttgttt aaagtttcac
     7921 catactttta aatcatgtgc tatttgaaaa gggctgttat gactggaatt aaatgttttt
     7981 ttttatatca gtgttttaga ttgaagtaca atatttatag gtaaaattca ccagcatatg
     8041 cttaaagata gccatgctcc aggtccttgt gtccttctaa ggtctgtttg tggtttaagc
     8101 tgtttgttga cacacgcttg tcatctccat ttttctgtga a
//
