LOCUS       sequence-204461-        7850 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 7850)
  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 7850)
  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-miR-182-dendra2
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             393..1085
                     /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    1196..1317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1233..1252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1287..1306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(1441..1628,1629..1640)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(1695..1715)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1695..1713)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1734..1750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1734..1750)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1747..1769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1758..1774
                     /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(1782..1788,1789..1806,1807..1812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1827..1848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1965..1982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2136..2724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2216..2235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2895..3686,3687..3755))
                     /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     3518..3537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3756..3860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4023..4044)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4440..4451,4452..4589)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      7017..7026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 agccaactgc tgttactctc gcccagaaat ctgcgccaac atggacacac ccagtggagc
       61 aagccatctg attttcatgg tcagattgta aagaagcaat tactcttgga cagtttaagc
      121 agaaaagcca ccaagtcaag tcactggtca tatgatgaac tcgaagatct aataaaagtt
      181 aatttaagcg ttcattacaa taaattcaag tgaatctgtt ttaagctagc ctttaacgtt
      241 tttaaattcc ttcaaacaaa ttagttcctc tttgcagttc ctcatttctc ggtgagctgg
      301 tgctttaatc cctttacttt ttcttcctct agggagtgat ggttggcatg ggacgaatta
      361 attcctgcag cccgggggta ccggtcgcca ccatgaacac cccgggaatt aacctgatca
      421 aggaggacat gcgcgtgaag gtgcacatgg agggcaacgt gaacggccac gccttcgtga
      481 tcgagggcga gggcaagggc aagccctacg agggcaccca gaccgccaac ctgaccgtga
      541 aggagggcgc ccccctgccc ttcagctacg acatcctgac caccgccgtg cactacggca
      601 accgggtgtt caccaagtac cccgaggaca tccccgacta cttcaagcag agcttccccg
      661 agggctacag ctgggagcgc accatgacct tcgaggacaa gggcatctgc accatccgca
      721 gcgacatcag cctggagggc gactgcttct tccagaacgt gcgcttcaag ggcaccaact
      781 tcccccccaa cggccccgtg atgcagaaga aaaccctgaa gtgggagccc agcaccgaga
      841 agctgcacgt gcgcgacggc ctgctggtgg gcaacatcaa catggccctg ctgctggagg
      901 gcggcggcca ctacctgtgc gacttcaaga ccacctacaa ggccaagaag gtggtgcagc
      961 tgcccgacgc ccacttcgtg gaccaccgca tcgagatcct gggcaacgac agcgactaca
     1021 acaaggtgaa gctgtacgag cacgccgtgg cccgctacag ccccctgccc agccaggtgt
     1081 ggtaatctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     1141 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     1201 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1261 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     1321 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     1381 cattttttaa ccaataggcc gaaatcggcg gatcctgcgg ccgcaaggga aaatagaatg
     1441 aagtgatctc caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag
     1501 tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta
     1561 ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac
     1621 tcaagtactt tacacctctg gttcttgacc ccctaccttc agcaagccca gcagatccac
     1681 tagatctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     1741 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1801 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1861 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3901 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3961 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4021 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4081 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4141 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4201 gacggggaaa gccggccaca atgacggcta ctacatggtg ccattccttc ctctttatag
     4261 gaatggagac tacctcctgt ccaacaatgc tcttggatac gagtacgcct acctgttgga
     4321 cccaggtcat tgcacaacac cagaaatgcc ctctgatctg caaaagacgt gaatatctgt
     4381 tcagacaccc atatccactc tgttccacac aggtcagagg tttgtccagg agttcttgac
     4441 agaggtgtaa aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa
     4501 ttttactcaa ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa
     4561 gtactccatt aaaattgtac ttgagtatta aggaagtaaa aggctagcaa gatctgctcg
     4621 agatacattt gattagaaga gtttgacttg ggttgattat agcaaatagt ggaacacaaa
     4681 cagattttga aatattcttg caaatttatt tattaaatcc attcaaatat tgtcattttt
     4741 tgtactgtgt aatttatatt gatttgctca tacaatattt acatttcgct taaacaatac
     4801 actattattt atatttaaaa atagctatat ataaatcaaa tgtatagctc ttttttacac
     4861 aattaatcaa agctaaaata tttgtcacac aaatacagta agccacttca caatgatatc
     4921 ttcaaaatct gtgcttgact gaatcatttc atttattgtt ttgtaattca cattatacct
     4981 tacgttctcc aggcacagtt tgaattgttg ataaatgaag ataatacatc tgatttaatt
     5041 agtctccttt gtttataata actttaatct aatttgatag taataattcc agcaatacat
     5101 ccattacaat aaaaatcgct tttttatttt gcacattagt ttgccggtat ttgtttaaag
     5161 tttcaccata cttttaaatc atgtgctatt tgaaaagggc tgttatgact ggaattaaat
     5221 gttttttttt atatcagtgt tttagattga agtacaatat ttataggtaa aattcaccag
     5281 catatgctta aagatagcca tgctccaggt ccttgtgtcc ttctaaggtc tgtttgtggt
     5341 ttaagctgtt tgttgacaca cgcttgtcat ctccattttt ctgtgaagtt attgtcagtt
     5401 tattgcacac tgggttgcat taagctgcag ttttttcaca acagttttcc acattgtaac
     5461 agtcagtctc caaattattc catcagtcct caaatttagt catttgaggc catgaaaaca
     5521 cttaaaacta catgaaggta gtttggcatt aggttttaga cttgttcttg tttggcatat
     5581 tgcaacctgt gtaaaaatga tttgattttt attttttttt tttttttttc agaaagaaac
     5641 ggtccaaaaa gagttatgtg aacatttatc catgtgttta atgtttttta aattaaaaaa
     5701 aaacattttt attaaaccac ttgtgactaa acctttttta gttattctat tttgtgacga
     5761 gtctgaatgg ccaaagtact cttgaatcca aaatctttat agtaaaatgt ttaatcttac
     5821 ttgcagtgtt gggtataatt tgtaacaaag ttgaattcca tatctgctga gtgaaaaagt
     5881 aaagtaagag attgctgtaa atatttaggt tatttattta ttaatacaat atagaaagta
     5941 acaaaacaca atgtaaaaaa tcaataaaga aagtaaatat taaaagtaat gataaactaa
     6001 tgatgtaaac aatattagca acaaagtttg tacaattcag ggagggtaaa gaatttcacc
     6061 taatagtcaa actctgtatt aaataatata aaatctttgt tcttagtctg aagaggagac
     6121 tgtgagagaa agcttactgg ttgttaaatt agaaaatttg taacttgcag attaaaatga
     6181 tttaaataat tttataaagg ccatgtttga aacaaatgta aaaacataat aaatgttaag
     6241 aatttcagat attgagattt tacaatttaa aaaaagcaat aaagtaatag cagagaccta
     6301 cactcttaaa gtaaattacc caactctgct tactagtaac ttttggaata gttactagta
     6361 tccattgaac aaatactaga atctaattaa attgtgtgag tgtccaatga aaagattctt
     6421 gtatttaaaa cagtaccagt atctatatat gtacaaaatt tagtgctagc aaaatgcaaa
     6481 taaatactac tgcatataat ggattgcatt gtaaatcgac tcgccataat acatttattt
     6541 cctattttta cttttaacaa aaaataagtt cactgagtat ttcatgtcat taaatcgcat
     6601 taaacctctt gcatgcttta tgtacactag tgcagttcgc taaatgacca ccggtgtcac
     6661 taataacacg atcgtttctg aattctgcat taaacgccat ttatacgttg ctcttattag
     6721 taaacaattt ctccatcatg agctcgacca gtattgcata tttcatcctc ttaaatccag
     6781 agttgtttct tttaatcact ttctcttcat gataaaagca gtttttggtt ttcagaagtt
     6841 tacaacaaac ccagtcagtt attgcctcag caaacactgg agcaactgca cacttgtgaa
     6901 aagcttcttg cgaaacacaa acctttctct ggacttataa ataaaggcat tcagcagctc
     6961 caccatacac ctccactggt cttgataaag cagatatcag cagtgataca ggtaccgcca
     7021 ccatggatga ggaaatcgct gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg
     7081 gttttgctgg agatgatgcc cctcgtgctg ttttcccctc cattgttgga cgacccagac
     7141 atcaggtaca tttaataaac catcctaata acatgttaat catattgaac cacattaaac
     7201 tctcatttgt gcattttaca tcttttaaaa ccgatttata ttcgttttta gataatgaag
     7261 tttattaaag tgttaattca cagctttatt gcttgttttg agaatctgct tgcataagtg
     7321 atttttaaac ttgatctgat tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat
     7381 cttcaattca cagccatgcc atagttttag gatacagtat aagtagacga attgggatgc
     7441 atcgtagatt ttatgctgtg tttgttatgg tttttcagtg gtagagattt tcagagtatt
     7501 attagggtta tagtcagtgt caggtttatt tatcgtcagc gtgatgggca aaaaacagct
     7561 ggcagtacgg gctcgatgtc tcaatgaatg gcccatttct tccactacta tttcaaggac
     7621 aaaaggttct ctgatggtat ttggcaatgg tagaactcac actggtgagg tagtcagatc
     7681 cggtggttct agacttgcca actactacct gagaacaact ccactgtaag gcatggatat
     7741 aaacaaatat agacaaagaa ctcagagcat catttcgaat atcaaggttg tttcaatgat
     7801 gtcacaagct ccgccccctg agcgagtgac tgtgagtagt gcaggttggg
//
