LOCUS       sequence-182766-        7938 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 7938)
  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 7938)
  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 730-Dendra2
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1741..1750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3055..3747
                     /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    3858..3979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3895..3914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3949..3968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4103..4290,4291..4302)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4357..4377)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4357..4375)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4396..4412)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4396..4412)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4409..4431)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4420..4436
                     /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(4444..4450,4451..4468,4469..4474))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4489..4510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4627..4644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4798..5386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4878..4897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5557..6348,6349..6417))
                     /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     6180..6199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6418..6522)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6685..6706)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7102..7113,7114..7251)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ccagcatatg cttaaagata gccatgctcc aggtccttgt gtccttctaa ggtctgtttg
       61 tggtttaagc tgtttgttga cacacgcttg tcatctccat ttttctgtga agttattgtc
      121 agtttattgc acactgggtt gcattaagct gcagtttttt cacaacagtt ttccacattg
      181 taacagtcag tctccaaatt attccatcag tcctcaaatt tagtcatttg aggccatgaa
      241 aacacttaaa actacatgaa ggtagtttgg cattaggttt tagacttgtt cttgtttggc
      301 atattgcaac ctgtgtaaaa atgatttgat ttttattttt tttttttttt tttcagaaag
      361 aaacggtcca aaaagagtta tgtgaacatt tatccatgtg tttaatgttt tttaaattaa
      421 aaaaaaacat ttttattaaa ccacttgtga ctaaaccttt tttagttatt ctattttgtg
      481 acgagtctga atggccaaag tactcttgaa tccaaaatct ttatagtaaa atgtttaatc
      541 ttacttgcag tgttgggtat aatttgtaac aaagttgaat tccatatctg ctgagtgaaa
      601 aagtaaagta agagattgct gtaaatattt aggttattta tttattaata caatatagaa
      661 agtaacaaaa cacaatgtaa aaaatcaata aagaaagtaa atattaaaag taatgataaa
      721 ctaatgatgt aaacaatatt agcaacaaag tttgtacaat tcagggaggg taaagaattt
      781 cacctaatag tcaaactctg tattaaataa tataaaatct ttgttcttag tctgaagagg
      841 agactgtgag agaaagctta ctggttgtta aattagaaaa tttgtaactt gcagattaaa
      901 atgatttaaa taattttata aaggccatgt ttgaaacaaa tgtaaaaaca taataaatgt
      961 taagaatttc agatattgag attttacaat ttaaaaaaag caataaagta atagcagaga
     1021 cctacactct taaagtaaat tacccaactc tgcttactag taacttttgg aatagttact
     1081 agtatccatt gaacaaatac tagaatctaa ttaaattgtg tgagtgtcca atgaaaagat
     1141 tcttgtattt aaaacagtac cagtatctat atatgtacaa aatttagtgc tagcaaaatg
     1201 caaataaata ctactgcata taatggattg cattgtaaat cgactcgcca taatacattt
     1261 atttcctatt tttactttta acaaaaaata agttcactga gtatttcatg tcattaaatc
     1321 gcattaaacc tcttgcatgc tttatgtaca ctagtgcagt tcgctaaatg accaccggtg
     1381 tcactaataa cacgatcgtt tctgaattct gcattaaacg ccatttatac gttgctctta
     1441 ttagtaaaca atttctccat catgagctcg accagtattg catatttcat cctcttaaat
     1501 ccagagttgt ttcttttaat cactttctct tcatgataaa agcagttttt ggttttcaga
     1561 agtttacaac aaacccagtc agttattgcc tcagcaaaca ctggagcaac tgcacacttg
     1621 tgaaaagctt cttgcgaaac acaaaccttt ctctggactt ataaataaag gcattcagca
     1681 gctccaccat acacctccac tggtcttgat aaagcagata tcagcagtga tacaggtacc
     1741 gccaccatgg atgaggaaat cgctgccctg gtcgttgaca acggctccgg tatgtgcaaa
     1801 gccggttttg ctggagatga tgcccctcgt gctgttttcc cctccattgt tggacgaccc
     1861 agacatcagg tacatttaat aaaccatcct aataacatgt taatcatatt gaaccacatt
     1921 aaactctcat ttgtgcattt tacatctttt aaaaccgatt tatattcgtt tttagataat
     1981 gaagtttatt aaagtgttaa ttcacagctt tattgcttgt tttgagaatc tgcttgcata
     2041 agtgattttt aaacttgatc tgattttcgt ttgtttcgaa gttcttcagc tttgaaagtc
     2101 agatcttcaa ttcacagcca tgccatagtt ttaggataca gtataagtag acgaattggg
     2161 atgcatcgta gattttatgc tgtgtttgtt atggtttttc agtggtagag attttcagag
     2221 tattattagg gttatagtca gtgtcaggtt tatttatcgt cagcgtgatg ggcaaaaaac
     2281 agctggcagt acgggctgtg accgcaggga agtcattaaa aatgaacccc cttttaaatt
     2341 tataaagtgt tccgagaaaa tgaccagtta aaatttcagt tcaaacgtaa tgcaattaaa
     2401 ctgcaatctc agtaccgagc aggcatccaa taaggggttc tccggtctcc tcattgtgca
     2461 tgctgtgtgt cttcagtctg gtcctcacag cgcctgcaat gtggaggcta ggggactcag
     2521 agacagcaga taaacaacac acacagagcc agtcgaacag cagccgctgc tgaagtcaaa
     2581 ggtatccatt atgttttatt ttgctgttaa tgtagcttgt ggaaaacacc tggtaattag
     2641 attgaagtga actcctgccc tcagccaact gctgttactc tcgcccagaa atctgcgcca
     2701 acatggacac acccagtgga gcaagccatc tgattttcat ggtcagattg taaagaagca
     2761 attactcttg gacagtttaa gcagaaaagc caccaagtca agtcactggt catatgatga
     2821 actcgaagat ctaataaaag ttaatttaag cgttcattac aataaattca agtgaatctg
     2881 ttttaagcta gcctttaacg tttttaaatt ccttcaaaca aattagttcc tctttgcagt
     2941 tcctcatttc tcggtgagct ggtgctttaa tccctttact ttttcttcct ctagggagtg
     3001 atggttggca tgggacgaat taattcctgc agcccggggg taccggtcgc caccatgaac
     3061 accccgggaa ttaacctgat caaggaggac atgcgcgtga aggtgcacat ggagggcaac
     3121 gtgaacggcc acgccttcgt gatcgagggc gagggcaagg gcaagcccta cgagggcacc
     3181 cagaccgcca acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg
     3241 accaccgccg tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac
     3301 tacttcaagc agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac
     3361 aagggcatct gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac
     3421 gtgcgcttca agggcaccaa cttccccccc aacggccccg tgatgcagaa gaaaaccctg
     3481 aagtgggagc ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc
     3541 aacatggccc tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac
     3601 aaggccaaga aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc
     3661 ctgggcaacg acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac
     3721 agccccctgc ccagccaggt gtggtaatct agatcataat cagccatacc acatttgtag
     3781 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3841 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3901 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3961 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4021 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg cggatcctgc
     4081 ggccgcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa
     4141 taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta
     4201 aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag
     4261 tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct
     4321 tcagcaagcc cagcagatcc actagatctt ttgttccctt tagtgagggt taattgcgcg
     4381 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4441 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4501 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4561 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4621 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4681 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4741 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4801 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4861 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4921 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4981 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5041 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5101 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5161 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5221 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5281 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5341 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5401 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5461 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5521 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5581 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5641 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5701 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5761 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5821 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5881 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5941 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6001 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6061 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6121 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6181 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6241 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6301 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6361 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6481 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6541 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     6601 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     6661 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6721 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6781 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6841 gggagccccc gatttagagc ttgacgggga aagccggcca caatgacggc tactacatgg
     6901 tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat gctcttggat
     6961 acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc
     7021 tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga
     7081 ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga
     7141 aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc
     7201 ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta
     7261 aaaggctagc aagatctgct cgagatacat ttgattagaa gagtttgact tgggttgatt
     7321 atagcaaata gtggaacaca aacagatttt gaaatattct tgcaaattta tttattaaat
     7381 ccattcaaat attgtcattt tttgtactgt gtaatttata ttgatttgct catacaatat
     7441 ttacatttcg cttaaacaat acactattat ttatatttaa aaatagctat atataaatca
     7501 aatgtatagc tcttttttac acaattaatc aaagctaaaa tatttgtcac acaaatacag
     7561 taagccactt cacaatgata tcttcaaaat ctgtgcttga ctgaatcatt tcatttattg
     7621 ttttgtaatt cacattatac cttacgttct ccaggcacag tttgaattgt tgataaatga
     7681 agataataca tctgatttaa ttagtctcct ttgtttataa taactttaat ctaatttgat
     7741 agtaataatt ccagcaatac atccattaca ataaaaatcg cttttttatt ttgcacatta
     7801 gtttgccggt atttgtttaa agtttcacca tacttttaaa tcatgtgcta tttgaaaagg
     7861 gctgttatga ctggaattaa atgttttttt ttatatcagt gttttagatt gaagtacaat
     7921 atttataggt aaaattca
//
