LOCUS       sequence-182598-        7973 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 7973)
  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 7973)
  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 107b-Dendra2
FEATURES             Location/Qualifiers
     source          1..7973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1766..1775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3115..3807
                     /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    3918..4039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3955..3974)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4009..4028
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4163..4350,4351..4362)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4417..4437)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4417..4435)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4456..4472)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4456..4472)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4469..4491)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4480..4496
                     /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(4504..4510,4511..4528,4529..4534))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4549..4570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4687..4704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4858..5446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4938..4957)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5617..6408,6409..6477))
                     /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     6240..6259
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6478..6582)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6745..6766)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7162..7173,7174..7311)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gtacaatatt tataggtaaa attcaccagc atatgcttaa agatagccat gctccaggtc
       61 cttgtgtcct tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc
      121 tccatttttc tgtgaagtta ttgtcagttt attgcacact gggttgcatt aagctgcagt
      181 tttttcacaa cagttttcca cattgtaaca gtcagtctcc aaattattcc atcagtcctc
      241 aaatttagtc atttgaggcc atgaaaacac ttaaaactac atgaaggtag tttggcatta
      301 ggttttagac ttgttcttgt ttggcatatt gcaacctgtg taaaaatgat ttgattttta
      361 tttttttttt ttttttttca gaaagaaacg gtccaaaaag agttatgtga acatttatcc
      421 atgtgtttaa tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa
      481 ccttttttag ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa
      541 aatctttata gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt
      601 tgaattccat atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt
      661 atttatttat taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa
      721 agtaaatatt aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt
      781 acaattcagg gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa
      841 aatctttgtt cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta
      901 gaaaatttgt aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa
      961 acaaatgtaa aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa
     1021 aaaagcaata aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt
     1081 actagtaact tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa
     1141 ttgtgtgagt gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg
     1201 tacaaaattt agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg
     1261 taaatcgact cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc
     1321 actgagtatt tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt
     1381 gcagttcgct aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt
     1441 aaacgccatt tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag
     1501 tattgcatat ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg
     1561 ataaaagcag tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc
     1621 aaacactgga gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg
     1681 gacttataaa taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc
     1741 agatatcagc agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt
     1801 tgacaacggc tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt
     1861 tttcccctcc attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa
     1921 catgttaatc atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac
     1981 cgatttatat tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg
     2041 cttgttttga gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt
     2101 tcgaagttct tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg
     2161 atacagtata agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt
     2221 ttttcagtgg tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt
     2281 atcgtcagcg tgatgggcaa aaaacagctg gcagtacggg cttcactaac aacatcggct
     2341 ccatcgcacg catgtgtgca gtcaatgagg tttgtagtgt tagtgtttgt tagtgtgcat
     2401 gtgttcaagt ggtgctcact gcagagccat ttgggcagag ctgagctcca gtgaggggct
     2461 catgctcccc ctggctggag cgcagctctg ctcaaatggc tctgcagcga gcagtgtctc
     2521 catgtgtttg catgtggagt gagcagcagt ctctctggcc actctgggct tctctacagt
     2581 gttgccttgt agcctggtga tcaagcagca ttgtacaggg ctttcagcgt gtacagaaca
     2641 ccatccgagc ggaaaccagc aacaacaccc agagaaaaca ggattcacat cacattagct
     2701 agcgtctgtg atgagatgat ggagccaact gctgttactc tcgcccagaa atctgcgcca
     2761 acatggacac acccagtgga gcaagccatc tgattttcat ggtcagattg taaagaagca
     2821 attactcttg gacagtttaa gcagaaaagc caccaagtca agtcactggt catatgatga
     2881 actcgaagat ctaataaaag ttaatttaag cgttcattac aataaattca agtgaatctg
     2941 ttttaagcta gcctttaacg tttttaaatt ccttcaaaca aattagttcc tctttgcagt
     3001 tcctcatttc tcggtgagct ggtgctttaa tccctttact ttttcttcct ctagggagtg
     3061 atggttggca tgggacgaat taattcctgc agcccggggg taccggtcgc caccatgaac
     3121 accccgggaa ttaacctgat caaggaggac atgcgcgtga aggtgcacat ggagggcaac
     3181 gtgaacggcc acgccttcgt gatcgagggc gagggcaagg gcaagcccta cgagggcacc
     3241 cagaccgcca acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg
     3301 accaccgccg tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac
     3361 tacttcaagc agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac
     3421 aagggcatct gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac
     3481 gtgcgcttca agggcaccaa cttccccccc aacggccccg tgatgcagaa gaaaaccctg
     3541 aagtgggagc ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc
     3601 aacatggccc tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac
     3661 aaggccaaga aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc
     3721 ctgggcaacg acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac
     3781 agccccctgc ccagccaggt gtggtaatct agatcataat cagccatacc acatttgtag
     3841 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3901 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3961 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4021 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4081 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg cggatcctgc
     4141 ggccgcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa
     4201 taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta
     4261 aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag
     4321 tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct
     4381 tcagcaagcc cagcagatcc actagatctt ttgttccctt tagtgagggt taattgcgcg
     4441 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4501 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4561 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4621 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4741 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4801 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4861 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4921 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4981 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5041 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5101 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5161 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5221 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5281 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5341 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5401 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5461 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5521 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5581 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5641 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5701 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5761 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5821 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5881 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5941 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6001 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6061 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6121 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6181 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6241 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6301 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6361 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6421 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6481 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6541 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6601 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     6661 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     6721 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6781 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6841 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6901 gggagccccc gatttagagc ttgacgggga aagccggcca caatgacggc tactacatgg
     6961 tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat gctcttggat
     7021 acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc
     7081 tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga
     7141 ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga
     7201 aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc
     7261 ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta
     7321 aaaggctagc aagatctgct cgagatacat ttgattagaa gagtttgact tgggttgatt
     7381 atagcaaata gtggaacaca aacagatttt gaaatattct tgcaaattta tttattaaat
     7441 ccattcaaat attgtcattt tttgtactgt gtaatttata ttgatttgct catacaatat
     7501 ttacatttcg cttaaacaat acactattat ttatatttaa aaatagctat atataaatca
     7561 aatgtatagc tcttttttac acaattaatc aaagctaaaa tatttgtcac acaaatacag
     7621 taagccactt cacaatgata tcttcaaaat ctgtgcttga ctgaatcatt tcatttattg
     7681 ttttgtaatt cacattatac cttacgttct ccaggcacag tttgaattgt tgataaatga
     7741 agataataca tctgatttaa ttagtctcct ttgtttataa taactttaat ctaatttgat
     7801 agtaataatt ccagcaatac atccattaca ataaaaatcg cttttttatt ttgcacatta
     7861 gtttgccggt atttgtttaa agtttcacca tacttttaaa tcatgtgcta tttgaaaagg
     7921 gctgttatga ctggaattaa atgttttttt ttatatcagt gttttagatt gaa
//
