LOCUS       sequence-182769-        7783 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 7783)
  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 7783)
  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 2184-Dendra2
FEATURES             Location/Qualifiers
     source          1..7783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1686..1695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2845..3537
                     /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    3648..3769
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3685..3704)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3739..3758
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3893..4080,4081..4092)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4147..4167)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4147..4165)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4186..4202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4186..4202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4199..4221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4210..4226
                     /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(4234..4240,4241..4258,4259..4264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4279..4300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4417..4434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4588..5176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4668..4687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5347..6138,6139..6207))
                     /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     5970..5989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6208..6312)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6475..6496)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6892..6903,6904..7041)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gtttgtggtt taagctgttt gttgacacac gcttgtcatc tccatttttc tgtgaagtta
       61 ttgtcagttt attgcacact gggttgcatt aagctgcagt tttttcacaa cagttttcca
      121 cattgtaaca gtcagtctcc aaattattcc atcagtcctc aaatttagtc atttgaggcc
      181 atgaaaacac ttaaaactac atgaaggtag tttggcatta ggttttagac ttgttcttgt
      241 ttggcatatt gcaacctgtg taaaaatgat ttgattttta tttttttttt ttttttttca
      301 gaaagaaacg gtccaaaaag agttatgtga acatttatcc atgtgtttaa tgttttttaa
      361 attaaaaaaa aacattttta ttaaaccact tgtgactaaa ccttttttag ttattctatt
      421 ttgtgacgag tctgaatggc caaagtactc ttgaatccaa aatctttata gtaaaatgtt
      481 taatcttact tgcagtgttg ggtataattt gtaacaaagt tgaattccat atctgctgag
      541 tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt atttatttat taatacaata
      601 tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa agtaaatatt aaaagtaatg
      661 ataaactaat gatgtaaaca atattagcaa caaagtttgt acaattcagg gagggtaaag
      721 aatttcacct aatagtcaaa ctctgtatta aataatataa aatctttgtt cttagtctga
      781 agaggagact gtgagagaaa gcttactggt tgttaaatta gaaaatttgt aacttgcaga
      841 ttaaaatgat ttaaataatt ttataaaggc catgtttgaa acaaatgtaa aaacataata
      901 aatgttaaga atttcagata ttgagatttt acaatttaaa aaaagcaata aagtaatagc
      961 agagacctac actcttaaag taaattaccc aactctgctt actagtaact tttggaatag
     1021 ttactagtat ccattgaaca aatactagaa tctaattaaa ttgtgtgagt gtccaatgaa
     1081 aagattcttg tatttaaaac agtaccagta tctatatatg tacaaaattt agtgctagca
     1141 aaatgcaaat aaatactact gcatataatg gattgcattg taaatcgact cgccataata
     1201 catttatttc ctatttttac ttttaacaaa aaataagttc actgagtatt tcatgtcatt
     1261 aaatcgcatt aaacctcttg catgctttat gtacactagt gcagttcgct aaatgaccac
     1321 cggtgtcact aataacacga tcgtttctga attctgcatt aaacgccatt tatacgttgc
     1381 tcttattagt aaacaatttc tccatcatga gctcgaccag tattgcatat ttcatcctct
     1441 taaatccaga gttgtttctt ttaatcactt tctcttcatg ataaaagcag tttttggttt
     1501 tcagaagttt acaacaaacc cagtcagtta ttgcctcagc aaacactgga gcaactgcac
     1561 acttgtgaaa agcttcttgc gaaacacaaa cctttctctg gacttataaa taaaggcatt
     1621 cagcagctcc accatacacc tccactggtc ttgataaagc agatatcagc agtgatacag
     1681 gtaccgccac catggatgag gaaatcgctg ccctggtcgt tgacaacggc tccggtatgt
     1741 gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt tttcccctcc attgttggac
     1801 gacccagaca tcaggtacat ttaataaacc atcctaataa catgttaatc atattgaacc
     1861 acattaaact ctcatttgtg cattttacat cttttaaaac cgatttatat tcgtttttag
     1921 ataatgaagt ttattaaagt gttaattcac agctttattg cttgttttga gaatctgctt
     1981 gcataagtga tttttaaact tgatctgatt ttcgtttgtt tcgaagttct tcagctttga
     2041 aagtcagatc ttcaattcac agccatgcca tagttttagg atacagtata agtagacgaa
     2101 ttgggatgca tcgtagattt tatgctgtgt ttgttatggt ttttcagtgg tagagatttt
     2161 cagagtatta ttagggttat agtcagtgtc aggtttattt atcgtcagcg tgatgggcaa
     2221 aaaacagctg gcagtacggg cttgtttttg cttcttttgc tgcttaaata gccgacattg
     2281 tctgttttgt aattaattat tcttagagat gttgtgtgtg tatgtgtgtt catctctgaa
     2341 tgatgcccta agccctaaac agtaagagtt tatgtgctga ggttgaaaat tcagcacatg
     2401 gtctcttact tgtagggaaa aaggtttcta tcgcctgtca cattgttttc gcagccaact
     2461 gctgttactc tcgcccagaa atctgcgcca acatggacac acccagtgga gcaagccatc
     2521 tgattttcat ggtcagattg taaagaagca attactcttg gacagtttaa gcagaaaagc
     2581 caccaagtca agtcactggt catatgatga actcgaagat ctaataaaag ttaatttaag
     2641 cgttcattac aataaattca agtgaatctg ttttaagcta gcctttaacg tttttaaatt
     2701 ccttcaaaca aattagttcc tctttgcagt tcctcatttc tcggtgagct ggtgctttaa
     2761 tccctttact ttttcttcct ctagggagtg atggttggca tgggacgaat taattcctgc
     2821 agcccggggg taccggtcgc caccatgaac accccgggaa ttaacctgat caaggaggac
     2881 atgcgcgtga aggtgcacat ggagggcaac gtgaacggcc acgccttcgt gatcgagggc
     2941 gagggcaagg gcaagcccta cgagggcacc cagaccgcca acctgaccgt gaaggagggc
     3001 gcccccctgc ccttcagcta cgacatcctg accaccgccg tgcactacgg caaccgggtg
     3061 ttcaccaagt accccgagga catccccgac tacttcaagc agagcttccc cgagggctac
     3121 agctgggagc gcaccatgac cttcgaggac aagggcatct gcaccatccg cagcgacatc
     3181 agcctggagg gcgactgctt cttccagaac gtgcgcttca agggcaccaa cttccccccc
     3241 aacggccccg tgatgcagaa gaaaaccctg aagtgggagc ccagcaccga gaagctgcac
     3301 gtgcgcgacg gcctgctggt gggcaacatc aacatggccc tgctgctgga gggcggcggc
     3361 cactacctgt gcgacttcaa gaccacctac aaggccaaga aggtggtgca gctgcccgac
     3421 gcccacttcg tggaccaccg catcgagatc ctgggcaacg acagcgacta caacaaggtg
     3481 aagctgtacg agcacgccgt ggcccgctac agccccctgc ccagccaggt gtggtaatct
     3541 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3601 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3661 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3721 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     3781 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3841 aaccaatagg ccgaaatcgg cggatcctgc ggccgcaagg gaaaatagaa tgaagtgatc
     3901 tccaaaaaat aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt
     3961 tttctaaaaa aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta
     4021 aagtaaaaat ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac
     4081 tttacacctc tggttcttga ccccctacct tcagcaagcc cagcagatcc actagatctt
     4141 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4201 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4261 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4321 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4381 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4441 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4501 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4561 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4621 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4681 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4741 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4801 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4861 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4921 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4981 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5041 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5101 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5161 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5221 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5281 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5341 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5401 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5461 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5521 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5581 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5641 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5701 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5761 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5821 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5881 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5941 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6001 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6061 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6121 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6181 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6241 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6301 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     6361 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     6421 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     6481 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6541 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6601 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6661 aagccggcca caatgacggc tactacatgg tgccattcct tcctctttat aggaatggag
     6721 actacctcct gtccaacaat gctcttggat acgagtacgc ctacctgttg gacccaggtc
     6781 attgcacaac accagaaatg ccctctgatc tgcaaaagac gtgaatatct gttcagacac
     6841 ccatatccac tctgttccac acaggtcaga ggtttgtcca ggagttcttg acagaggtgt
     6901 aaaaagtact caaaaatttt actcaagtga aagtacaagt acttagggaa aattttactc
     6961 aattaaaagt aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa aagtactcca
     7021 ttaaaattgt acttgagtat taaggaagta aaaggctagc aagatctgct cgagatacat
     7081 ttgattagaa gagtttgact tgggttgatt atagcaaata gtggaacaca aacagatttt
     7141 gaaatattct tgcaaattta tttattaaat ccattcaaat attgtcattt tttgtactgt
     7201 gtaatttata ttgatttgct catacaatat ttacatttcg cttaaacaat acactattat
     7261 ttatatttaa aaatagctat atataaatca aatgtatagc tcttttttac acaattaatc
     7321 aaagctaaaa tatttgtcac acaaatacag taagccactt cacaatgata tcttcaaaat
     7381 ctgtgcttga ctgaatcatt tcatttattg ttttgtaatt cacattatac cttacgttct
     7441 ccaggcacag tttgaattgt tgataaatga agataataca tctgatttaa ttagtctcct
     7501 ttgtttataa taactttaat ctaatttgat agtaataatt ccagcaatac atccattaca
     7561 ataaaaatcg cttttttatt ttgcacatta gtttgccggt atttgtttaa agtttcacca
     7621 tacttttaaa tcatgtgcta tttgaaaagg gctgttatga ctggaattaa atgttttttt
     7681 ttatatcagt gttttagatt gaagtacaat atttataggt aaaattcacc agcatatgct
     7741 taaagatagc catgctccag gtccttgtgt ccttctaagg tct
//
