LOCUS       sequence-182669-        8223 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 8223)
  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 8223)
  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 455a-Dendra2
FEATURES             Location/Qualifiers
     source          1..8223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1696..1705
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3295..3987
                     /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    4098..4219
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4135..4154)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4189..4208
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4343..4530,4531..4542)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4597..4617)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4597..4615)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4636..4652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4636..4652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4649..4671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4660..4676
                     /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(4684..4690,4691..4708,4709..4714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4729..4750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4867..4884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5038..5626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5118..5137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5797..6588,6589..6657))
                     /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     6420..6439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6658..6762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6925..6946)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7342..7353,7354..7491)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc tccatttttc
       61 tgtgaagtta ttgtcagttt attgcacact gggttgcatt aagctgcagt tttttcacaa
      121 cagttttcca cattgtaaca gtcagtctcc aaattattcc atcagtcctc aaatttagtc
      181 atttgaggcc atgaaaacac ttaaaactac atgaaggtag tttggcatta ggttttagac
      241 ttgttcttgt ttggcatatt gcaacctgtg taaaaatgat ttgattttta tttttttttt
      301 ttttttttca gaaagaaacg gtccaaaaag agttatgtga acatttatcc atgtgtttaa
      361 tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa ccttttttag
      421 ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa aatctttata
      481 gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt tgaattccat
      541 atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt atttatttat
      601 taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa agtaaatatt
      661 aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt acaattcagg
      721 gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa aatctttgtt
      781 cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta gaaaatttgt
      841 aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa acaaatgtaa
      901 aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa aaaagcaata
      961 aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt actagtaact
     1021 tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa ttgtgtgagt
     1081 gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg tacaaaattt
     1141 agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg taaatcgact
     1201 cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc actgagtatt
     1261 tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt gcagttcgct
     1321 aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt aaacgccatt
     1381 tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag tattgcatat
     1441 ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg ataaaagcag
     1501 tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc aaacactgga
     1561 gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg gacttataaa
     1621 taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc agatatcagc
     1681 agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt tgacaacggc
     1741 tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt tttcccctcc
     1801 attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa catgttaatc
     1861 atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac cgatttatat
     1921 tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg cttgttttga
     1981 gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt tcgaagttct
     2041 tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg atacagtata
     2101 agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt ttttcagtgg
     2161 tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt atcgtcagcg
     2221 tgatgggcaa aaaacagctg gcagtacggg ctaccgaaga tgcctgggat tccaatgaag
     2281 ccctaaaaca ggggagtgta agaacacaag aaacaagcag acagacttta ggctgattct
     2341 gatccaaaca ccacttgctg aacttgcaga ctgacatgaa aaaggtcgtt ttgaggacag
     2401 cggtcgctcg gtgaagatct aggccttgag gcaagtgtat atgcccatgg actgcatggt
     2461 gctggcttcc acgatgtagt ccaagggcac ataccctcac accagggatc acacgggcat
     2521 tcggcaggat ggctaagggt gggtcacaga gttaatgcag atgaacatac aatgagatct
     2581 tgaagagaaa aagacatgag tgatgagtca atgatgcact ttatggattg atgatgatga
     2641 tgataataaa aagcaatatg agaattcaat ggggtatatg cacacagact tttaattttc
     2701 agtcagccaa cccaatcgct tctggttaat tgtcgtgcca ttacaaaatc tgatttcttt
     2761 aaaaaccttt cgtctcattt tacgcttgaa caactaaatg aatgcttaaa gcctcattcg
     2821 cactacaagt gacttgcagc ggcaaagcga caagtgacca ttcattttaa tggagagcga
     2881 gcgacttctg gcgacctctg tctaccaact gctgttactc tcgcccagaa atctgcgcca
     2941 acatggacac acccagtgga gcaagccatc tgattttcat ggtcagattg taaagaagca
     3001 attactcttg gacagtttaa gcagaaaagc caccaagtca agtcactggt catatgatga
     3061 actcgaagat ctaataaaag ttaatttaag cgttcattac aataaattca agtgaatctg
     3121 ttttaagcta gcctttaacg tttttaaatt ccttcaaaca aattagttcc tctttgcagt
     3181 tcctcatttc tcggtgagct ggtgctttaa tccctttact ttttcttcct ctagggagtg
     3241 atggttggca tgggacgaat taattcctgc agcccggggg taccggtcgc caccatgaac
     3301 accccgggaa ttaacctgat caaggaggac atgcgcgtga aggtgcacat ggagggcaac
     3361 gtgaacggcc acgccttcgt gatcgagggc gagggcaagg gcaagcccta cgagggcacc
     3421 cagaccgcca acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg
     3481 accaccgccg tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac
     3541 tacttcaagc agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac
     3601 aagggcatct gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac
     3661 gtgcgcttca agggcaccaa cttccccccc aacggccccg tgatgcagaa gaaaaccctg
     3721 aagtgggagc ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc
     3781 aacatggccc tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac
     3841 aaggccaaga aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc
     3901 ctgggcaacg acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac
     3961 agccccctgc ccagccaggt gtggtaatct agatcataat cagccatacc acatttgtag
     4021 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     4081 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4141 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4201 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4261 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg cggatcctgc
     4321 ggccgcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa
     4381 taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta
     4441 aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag
     4501 tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct
     4561 tcagcaagcc cagcagatcc actagatctt ttgttccctt tagtgagggt taattgcgcg
     4621 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4681 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4741 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4801 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4861 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4921 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4981 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5461 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5641 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6481 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6781 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     6841 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     6901 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6961 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     7021 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     7081 gggagccccc gatttagagc ttgacgggga aagccggcca caatgacggc tactacatgg
     7141 tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat gctcttggat
     7201 acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc
     7261 tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga
     7321 ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga
     7381 aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc
     7441 ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta
     7501 aaaggctagc aagatctgct cgagatacat ttgattagaa gagtttgact tgggttgatt
     7561 atagcaaata gtggaacaca aacagatttt gaaatattct tgcaaattta tttattaaat
     7621 ccattcaaat attgtcattt tttgtactgt gtaatttata ttgatttgct catacaatat
     7681 ttacatttcg cttaaacaat acactattat ttatatttaa aaatagctat atataaatca
     7741 aatgtatagc tcttttttac acaattaatc aaagctaaaa tatttgtcac acaaatacag
     7801 taagccactt cacaatgata tcttcaaaat ctgtgcttga ctgaatcatt tcatttattg
     7861 ttttgtaatt cacattatac cttacgttct ccaggcacag tttgaattgt tgataaatga
     7921 agataataca tctgatttaa ttagtctcct ttgtttataa taactttaat ctaatttgat
     7981 agtaataatt ccagcaatac atccattaca ataaaaatcg cttttttatt ttgcacatta
     8041 gtttgccggt atttgtttaa agtttcacca tacttttaaa tcatgtgcta tttgaaaagg
     8101 gctgttatga ctggaattaa atgttttttt ttatatcagt gttttagatt gaagtacaat
     8161 atttataggt aaaattcacc agcatatgct taaagatagc catgctccag gtccttgtgt
     8221 cct
//
