LOCUS       sequence-182649-        7959 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 7959)
  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 7959)
  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 200b-Dendra2
FEATURES             Location/Qualifiers
     source          1..7959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1346..1355
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2681..3373
                     /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    3484..3605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3521..3540)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3575..3594
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3729..3916,3917..3928)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3983..4003)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3983..4001)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4022..4038)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4022..4038)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4035..4057)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4046..4062
                     /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(4070..4076,4077..4094,4095..4100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4115..4136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4253..4270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4424..5012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4504..4523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5183..5974,5975..6043))
                     /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     5806..5825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6044..6148)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6311..6332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6728..6739,6740..6877)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 atgtgtttaa tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa
       61 ccttttttag ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa
      121 aatctttata gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt
      181 tgaattccat atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt
      241 atttatttat taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa
      301 agtaaatatt aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt
      361 acaattcagg gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa
      421 aatctttgtt cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta
      481 gaaaatttgt aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa
      541 acaaatgtaa aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa
      601 aaaagcaata aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt
      661 actagtaact tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa
      721 ttgtgtgagt gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg
      781 tacaaaattt agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg
      841 taaatcgact cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc
      901 actgagtatt tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt
      961 gcagttcgct aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt
     1021 aaacgccatt tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag
     1081 tattgcatat ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg
     1141 ataaaagcag tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc
     1201 aaacactgga gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg
     1261 gacttataaa taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc
     1321 agatatcagc agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt
     1381 tgacaacggc tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt
     1441 tttcccctcc attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa
     1501 catgttaatc atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac
     1561 cgatttatat tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg
     1621 cttgttttga gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt
     1681 tcgaagttct tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg
     1741 atacagtata agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt
     1801 ttttcagtgg tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt
     1861 atcgtcagcg tgatgggcaa aaaacagctg gcagtacggg ctgcaggttg ttttaccggc
     1921 ataccccttt gtctaggaca gttgactgag tcagagctgt tatcaagtcc tgtttattct
     1981 aagtgcagtt tctgtggttg actccccaag tgatgcttac tatccaactt taaacaccct
     2041 aataattaat atcttgcaat actgttaata ttatgtgaag cattattact ttgagacttt
     2101 gtgttgtttg ataagacctc tgattttggt agtcgtctcc atcttacgag gcagcattgg
     2161 atttcattac tttttctaat actgcctggt aatgatgatg attgctgcct acaagctaca
     2221 gatcaagatc attcctggga aaataaaccc tcatcactgg cacttagcag ccatcttacc
     2281 ggacagtgag ccaactgctg ttactctcgc ccagaaatct gcgccaacat ggacacaccc
     2341 agtggagcaa gccatctgat tttcatggtc agattgtaaa gaagcaatta ctcttggaca
     2401 gtttaagcag aaaagccacc aagtcaagtc actggtcata tgatgaactc gaagatctaa
     2461 taaaagttaa tttaagcgtt cattacaata aattcaagtg aatctgtttt aagctagcct
     2521 ttaacgtttt taaattcctt caaacaaatt agttcctctt tgcagttcct catttctcgg
     2581 tgagctggtg ctttaatccc tttacttttt cttcctctag ggagtgatgg ttggcatggg
     2641 acgaattaat tcctgcagcc cgggggtacc ggtcgccacc atgaacaccc cgggaattaa
     2701 cctgatcaag gaggacatgc gcgtgaaggt gcacatggag ggcaacgtga acggccacgc
     2761 cttcgtgatc gagggcgagg gcaagggcaa gccctacgag ggcacccaga ccgccaacct
     2821 gaccgtgaag gagggcgccc ccctgccctt cagctacgac atcctgacca ccgccgtgca
     2881 ctacggcaac cgggtgttca ccaagtaccc cgaggacatc cccgactact tcaagcagag
     2941 cttccccgag ggctacagct gggagcgcac catgaccttc gaggacaagg gcatctgcac
     3001 catccgcagc gacatcagcc tggagggcga ctgcttcttc cagaacgtgc gcttcaaggg
     3061 caccaacttc ccccccaacg gccccgtgat gcagaagaaa accctgaagt gggagcccag
     3121 caccgagaag ctgcacgtgc gcgacggcct gctggtgggc aacatcaaca tggccctgct
     3181 gctggagggc ggcggccact acctgtgcga cttcaagacc acctacaagg ccaagaaggt
     3241 ggtgcagctg cccgacgccc acttcgtgga ccaccgcatc gagatcctgg gcaacgacag
     3301 cgactacaac aaggtgaagc tgtacgagca cgccgtggcc cgctacagcc ccctgcccag
     3361 ccaggtgtgg taatctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     3421 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3481 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3601 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3661 aatcagctca ttttttaacc aataggccga aatcggcgga tcctgcggcc gcaagggaaa
     3721 atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga
     3781 gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt
     3841 taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt
     3901 aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag caagcccagc
     3961 agatccacta gatcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     4021 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4081 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4141 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4201 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4261 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4321 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4381 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4441 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4501 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4561 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4621 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4681 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4741 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4801 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4861 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4921 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4981 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5041 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5101 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5161 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5221 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5281 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5341 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5401 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5461 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5521 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5581 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5641 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5701 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5761 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5821 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5881 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5941 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6001 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6061 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6121 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     6181 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6241 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6301 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     6361 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     6421 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     6481 tagagcttga cggggaaagc cggccacaat gacggctact acatggtgcc attccttcct
     6541 ctttatagga atggagacta cctcctgtcc aacaatgctc ttggatacga gtacgcctac
     6601 ctgttggacc caggtcattg cacaacacca gaaatgccct ctgatctgca aaagacgtga
     6661 atatctgttc agacacccat atccactctg ttccacacag gtcagaggtt tgtccaggag
     6721 ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt acaagtactt
     6781 agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa
     6841 gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag gctagcaaga
     6901 tctgctcgag atacatttga ttagaagagt ttgacttggg ttgattatag caaatagtgg
     6961 aacacaaaca gattttgaaa tattcttgca aatttattta ttaaatccat tcaaatattg
     7021 tcattttttg tactgtgtaa tttatattga tttgctcata caatatttac atttcgctta
     7081 aacaatacac tattatttat atttaaaaat agctatatat aaatcaaatg tatagctctt
     7141 ttttacacaa ttaatcaaag ctaaaatatt tgtcacacaa atacagtaag ccacttcaca
     7201 atgatatctt caaaatctgt gcttgactga atcatttcat ttattgtttt gtaattcaca
     7261 ttatacctta cgttctccag gcacagtttg aattgttgat aaatgaagat aatacatctg
     7321 atttaattag tctcctttgt ttataataac tttaatctaa tttgatagta ataattccag
     7381 caatacatcc attacaataa aaatcgcttt tttattttgc acattagttt gccggtattt
     7441 gtttaaagtt tcaccatact tttaaatcat gtgctatttg aaaagggctg ttatgactgg
     7501 aattaaatgt ttttttttat atcagtgttt tagattgaag tacaatattt ataggtaaaa
     7561 ttcaccagca tatgcttaaa gatagccatg ctccaggtcc ttgtgtcctt ctaaggtctg
     7621 tttgtggttt aagctgtttg ttgacacacg cttgtcatct ccatttttct gtgaagttat
     7681 tgtcagttta ttgcacactg ggttgcatta agctgcagtt ttttcacaac agttttccac
     7741 attgtaacag tcagtctcca aattattcca tcagtcctca aatttagtca tttgaggcca
     7801 tgaaaacact taaaactaca tgaaggtagt ttggcattag gttttagact tgttcttgtt
     7861 tggcatattg caacctgtgt aaaaatgatt tgatttttat tttttttttt tttttttcag
     7921 aaagaaacgg tccaaaaaga gttatgtgaa catttatcc
//
