LOCUS       sequence-182640-        7950 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 7950)
  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 7950)
  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 148-Dendra2
FEATURES             Location/Qualifiers
     source          1..7950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(105..126)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(522..533,534..671)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      3099..3108
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4425..5117
                     /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    5228..5349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5265..5284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5319..5338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(5473..5660,5661..5672)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5727..5747)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5727..5745)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5766..5782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5766..5782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5779..5801)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5790..5806
                     /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(5814..5820,5821..5838,5839..5844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5859..5880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5997..6014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6168..6756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6248..6267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6927..7718,7719..7787))
                     /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     7550..7569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7788..7892)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
       61 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
      121 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
      181 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
      241 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcca
      301 caatgacggc tactacatgg tgccattcct tcctctttat aggaatggag actacctcct
      361 gtccaacaat gctcttggat acgagtacgc ctacctgttg gacccaggtc attgcacaac
      421 accagaaatg ccctctgatc tgcaaaagac gtgaatatct gttcagacac ccatatccac
      481 tctgttccac acaggtcaga ggtttgtcca ggagttcttg acagaggtgt aaaaagtact
      541 caaaaatttt actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt
      601 aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt
      661 acttgagtat taaggaagta aaaggctagc aagatctgct cgagatacat ttgattagaa
      721 gagtttgact tgggttgatt atagcaaata gtggaacaca aacagatttt gaaatattct
      781 tgcaaattta tttattaaat ccattcaaat attgtcattt tttgtactgt gtaatttata
      841 ttgatttgct catacaatat ttacatttcg cttaaacaat acactattat ttatatttaa
      901 aaatagctat atataaatca aatgtatagc tcttttttac acaattaatc aaagctaaaa
      961 tatttgtcac acaaatacag taagccactt cacaatgata tcttcaaaat ctgtgcttga
     1021 ctgaatcatt tcatttattg ttttgtaatt cacattatac cttacgttct ccaggcacag
     1081 tttgaattgt tgataaatga agataataca tctgatttaa ttagtctcct ttgtttataa
     1141 taactttaat ctaatttgat agtaataatt ccagcaatac atccattaca ataaaaatcg
     1201 cttttttatt ttgcacatta gtttgccggt atttgtttaa agtttcacca tacttttaaa
     1261 tcatgtgcta tttgaaaagg gctgttatga ctggaattaa atgttttttt ttatatcagt
     1321 gttttagatt gaagtacaat atttataggt aaaattcacc agcatatgct taaagatagc
     1381 catgctccag gtccttgtgt ccttctaagg tctgtttgtg gtttaagctg tttgttgaca
     1441 cacgcttgtc atctccattt ttctgtgaag ttattgtcag tttattgcac actgggttgc
     1501 attaagctgc agttttttca caacagtttt ccacattgta acagtcagtc tccaaattat
     1561 tccatcagtc ctcaaattta gtcatttgag gccatgaaaa cacttaaaac tacatgaagg
     1621 tagtttggca ttaggtttta gacttgttct tgtttggcat attgcaacct gtgtaaaaat
     1681 gatttgattt ttattttttt tttttttttt tcagaaagaa acggtccaaa aagagttatg
     1741 tgaacattta tccatgtgtt taatgttttt taaattaaaa aaaaacattt ttattaaacc
     1801 acttgtgact aaaccttttt tagttattct attttgtgac gagtctgaat ggccaaagta
     1861 ctcttgaatc caaaatcttt atagtaaaat gtttaatctt acttgcagtg ttgggtataa
     1921 tttgtaacaa agttgaattc catatctgct gagtgaaaaa gtaaagtaag agattgctgt
     1981 aaatatttag gttatttatt tattaataca atatagaaag taacaaaaca caatgtaaaa
     2041 aatcaataaa gaaagtaaat attaaaagta atgataaact aatgatgtaa acaatattag
     2101 caacaaagtt tgtacaattc agggagggta aagaatttca cctaatagtc aaactctgta
     2161 ttaaataata taaaatcttt gttcttagtc tgaagaggag actgtgagag aaagcttact
     2221 ggttgttaaa ttagaaaatt tgtaacttgc agattaaaat gatttaaata attttataaa
     2281 ggccatgttt gaaacaaatg taaaaacata ataaatgtta agaatttcag atattgagat
     2341 tttacaattt aaaaaaagca ataaagtaat agcagagacc tacactctta aagtaaatta
     2401 cccaactctg cttactagta acttttggaa tagttactag tatccattga acaaatacta
     2461 gaatctaatt aaattgtgtg agtgtccaat gaaaagattc ttgtatttaa aacagtacca
     2521 gtatctatat atgtacaaaa tttagtgcta gcaaaatgca aataaatact actgcatata
     2581 atggattgca ttgtaaatcg actcgccata atacatttat ttcctatttt tacttttaac
     2641 aaaaaataag ttcactgagt atttcatgtc attaaatcgc attaaacctc ttgcatgctt
     2701 tatgtacact agtgcagttc gctaaatgac caccggtgtc actaataaca cgatcgtttc
     2761 tgaattctgc attaaacgcc atttatacgt tgctcttatt agtaaacaat ttctccatca
     2821 tgagctcgac cagtattgca tatttcatcc tcttaaatcc agagttgttt cttttaatca
     2881 ctttctcttc atgataaaag cagtttttgg ttttcagaag tttacaacaa acccagtcag
     2941 ttattgcctc agcaaacact ggagcaactg cacacttgtg aaaagcttct tgcgaaacac
     3001 aaacctttct ctggacttat aaataaaggc attcagcagc tccaccatac acctccactg
     3061 gtcttgataa agcagatatc agcagtgata caggtaccgc caccatggat gaggaaatcg
     3121 ctgccctggt cgttgacaac ggctccggta tgtgcaaagc cggttttgct ggagatgatg
     3181 cccctcgtgc tgttttcccc tccattgttg gacgacccag acatcaggta catttaataa
     3241 accatcctaa taacatgtta atcatattga accacattaa actctcattt gtgcatttta
     3301 catcttttaa aaccgattta tattcgtttt tagataatga agtttattaa agtgttaatt
     3361 cacagcttta ttgcttgttt tgagaatctg cttgcataag tgatttttaa acttgatctg
     3421 attttcgttt gtttcgaagt tcttcagctt tgaaagtcag atcttcaatt cacagccatg
     3481 ccatagtttt aggatacagt ataagtagac gaattgggat gcatcgtaga ttttatgctg
     3541 tgtttgttat ggtttttcag tggtagagat tttcagagta ttattagggt tatagtcagt
     3601 gtcaggttta tttatcgtca gcgtgatggg caaaaaacag ctggcagtac gggctaaggg
     3661 aacgagcatg agatggtggt aattgtaatg atgttttgtg tgtgttgtcg aggaacacga
     3721 cgtcgttttc atgcttttct cctccaggcc tgagctttaa actcccaaaa caaagttctg
     3781 taatgcactg actgcaaaca ttcagagtcg gagtgtatca cagaacttta cttggaaagc
     3841 cagagagctg cttgatgata ttcgcactct tccgccatct gcaagaaaca aacacacacg
     3901 ttacattact ctcctctgtc tcgcatatta cactacatac ttactcaaaa aatactgcac
     3961 tgtcgaaaac caaatttaca agaacaatag attaatgctt ggtttgtggt gttattctga
     4021 agcgtgttgg caagccaact gctgttactc tcgcccagaa atctgcgcca acatggacac
     4081 acccagtgga gcaagccatc tgattttcat ggtcagattg taaagaagca attactcttg
     4141 gacagtttaa gcagaaaagc caccaagtca agtcactggt catatgatga actcgaagat
     4201 ctaataaaag ttaatttaag cgttcattac aataaattca agtgaatctg ttttaagcta
     4261 gcctttaacg tttttaaatt ccttcaaaca aattagttcc tctttgcagt tcctcatttc
     4321 tcggtgagct ggtgctttaa tccctttact ttttcttcct ctagggagtg atggttggca
     4381 tgggacgaat taattcctgc agcccggggg taccggtcgc caccatgaac accccgggaa
     4441 ttaacctgat caaggaggac atgcgcgtga aggtgcacat ggagggcaac gtgaacggcc
     4501 acgccttcgt gatcgagggc gagggcaagg gcaagcccta cgagggcacc cagaccgcca
     4561 acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg accaccgccg
     4621 tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac tacttcaagc
     4681 agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac aagggcatct
     4741 gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac gtgcgcttca
     4801 agggcaccaa cttccccccc aacggccccg tgatgcagaa gaaaaccctg aagtgggagc
     4861 ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc aacatggccc
     4921 tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac aaggccaaga
     4981 aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc ctgggcaacg
     5041 acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac agccccctgc
     5101 ccagccaggt gtggtaatct agatcataat cagccatacc acatttgtag aggttttact
     5161 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     5221 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5281 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5341 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5401 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg cggatcctgc ggccgcaagg
     5461 gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa taaaattgta
     5521 aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta aaagtattga
     5581 tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag tacagtaatc
     5641 aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct tcagcaagcc
     5701 cagcagatcc actagatctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     5761 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5821 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5881 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5941 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6001 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6061 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     7921 ttgtaagcgt taatattttg ttaaaattcg
//
