LOCUS       sequence-182573-        7936 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 7936)
  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 7936)
  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 Let 7-2-dendra2
FEATURES             Location/Qualifiers
     source          1..7936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1733..1742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3045..3737
                     /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    3848..3969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3885..3904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3939..3958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4093..4280,4281..4292)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4347..4367)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4347..4365)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4386..4402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4386..4402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4399..4421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4410..4426
                     /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(4434..4440,4441..4458,4459..4464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4479..4500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4617..4634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4788..5376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4868..4887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5547..6338,6339..6407))
                     /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     6170..6189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6408..6512)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6675..6696)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7092..7103,7104..7241)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tgcttaaaga tagccatgct ccaggtcctt gtgtccttct aaggtctgtt tgtggtttaa
       61 gctgtttgtt gacacacgct tgtcatctcc atttttctgt gaagttattg tcagtttatt
      121 gcacactggg ttgcattaag ctgcagtttt ttcacaacag ttttccacat tgtaacagtc
      181 agtctccaaa ttattccatc agtcctcaaa tttagtcatt tgaggccatg aaaacactta
      241 aaactacatg aaggtagttt ggcattaggt tttagacttg ttcttgtttg gcatattgca
      301 acctgtgtaa aaatgatttg atttttattt tttttttttt tttttcagaa agaaacggtc
      361 caaaaagagt tatgtgaaca tttatccatg tgtttaatgt tttttaaatt aaaaaaaaac
      421 atttttatta aaccacttgt gactaaacct tttttagtta ttctattttg tgacgagtct
      481 gaatggccaa agtactcttg aatccaaaat ctttatagta aaatgtttaa tcttacttgc
      541 agtgttgggt ataatttgta acaaagttga attccatatc tgctgagtga aaaagtaaag
      601 taagagattg ctgtaaatat ttaggttatt tatttattaa tacaatatag aaagtaacaa
      661 aacacaatgt aaaaaatcaa taaagaaagt aaatattaaa agtaatgata aactaatgat
      721 gtaaacaata ttagcaacaa agtttgtaca attcagggag ggtaaagaat ttcacctaat
      781 agtcaaactc tgtattaaat aatataaaat ctttgttctt agtctgaaga ggagactgtg
      841 agagaaagct tactggttgt taaattagaa aatttgtaac ttgcagatta aaatgattta
      901 aataatttta taaaggccat gtttgaaaca aatgtaaaaa cataataaat gttaagaatt
      961 tcagatattg agattttaca atttaaaaaa agcaataaag taatagcaga gacctacact
     1021 cttaaagtaa attacccaac tctgcttact agtaactttt ggaatagtta ctagtatcca
     1081 ttgaacaaat actagaatct aattaaattg tgtgagtgtc caatgaaaag attcttgtat
     1141 ttaaaacagt accagtatct atatatgtac aaaatttagt gctagcaaaa tgcaaataaa
     1201 tactactgca tataatggat tgcattgtaa atcgactcgc cataatacat ttatttccta
     1261 tttttacttt taacaaaaaa taagttcact gagtatttca tgtcattaaa tcgcattaaa
     1321 cctcttgcat gctttatgta cactagtgca gttcgctaaa tgaccaccgg tgtcactaat
     1381 aacacgatcg tttctgaatt ctgcattaaa cgccatttat acgttgctct tattagtaaa
     1441 caatttctcc atcatgagct cgaccagtat tgcatatttc atcctcttaa atccagagtt
     1501 gtttctttta atcactttct cttcatgata aaagcagttt ttggttttca gaagtttaca
     1561 acaaacccag tcagttattg cctcagcaaa cactggagca actgcacact tgtgaaaagc
     1621 ttcttgcgaa acacaaacct ttctctggac ttataaataa aggcattcag cagctccacc
     1681 atacacctcc actggtcttg ataaagcaga tatcagcagt gatacaggta ccgccaccat
     1741 ggatgaggaa atcgctgccc tggtcgttga caacggctcc ggtatgtgca aagccggttt
     1801 tgctggagat gatgcccctc gtgctgtttt cccctccatt gttggacgac ccagacatca
     1861 ggtacattta ataaaccatc ctaataacat gttaatcata ttgaaccaca ttaaactctc
     1921 atttgtgcat tttacatctt ttaaaaccga tttatattcg tttttagata atgaagttta
     1981 ttaaagtgtt aattcacagc tttattgctt gttttgagaa tctgcttgca taagtgattt
     2041 ttaaacttga tctgattttc gtttgtttcg aagttcttca gctttgaaag tcagatcttc
     2101 aattcacagc catgccatag ttttaggata cagtataagt agacgaattg ggatgcatcg
     2161 tagattttat gctgtgtttg ttatggtttt tcagtggtag agattttcag agtattatta
     2221 gggttatagt cagtgtcagg tttatttatc gtcagcgtga tgggcaaaaa acagctggca
     2281 gtacgggctg ttgccttaca ctggttgtcc agtaggggaa gacattacac ttgaaatata
     2341 ttgacacctg ctagtgctag tgatgtagga atggatactg gagtctgtgc gctgcaggct
     2401 gaggtagttg gttgtatggt tttgcatcat aatcagcctg gagttaactg tacaaccttc
     2461 tagctttccc tgcggtgtca ccattacgtc gaggtgagta tgaacagctg ccatatagac
     2521 gagcatttca agtgtttcca ccttataatg gaggatagaa gtagtttcat tgttgttgtt
     2581 gtattttgtt tcggtctttg ttgtgctgtc gttaactggc tgttagtcag tccgtctgcc
     2641 aaatggaggg ttagccaact gctgttactc tcgcccagaa atctgcgcca acatggacac
     2701 acccagtgga gcaagccatc tgattttcat ggtcagattg taaagaagca attactcttg
     2761 gacagtttaa gcagaaaagc caccaagtca agtcactggt catatgatga actcgaagat
     2821 ctaataaaag ttaatttaag cgttcattac aataaattca agtgaatctg ttttaagcta
     2881 gcctttaacg tttttaaatt ccttcaaaca aattagttcc tctttgcagt tcctcatttc
     2941 tcggtgagct ggtgctttaa tccctttact ttttcttcct ctagggagtg atggttggca
     3001 tgggacgaat taattcctgc agcccggggg taccggtcgc caccatgaac accccgggaa
     3061 ttaacctgat caaggaggac atgcgcgtga aggtgcacat ggagggcaac gtgaacggcc
     3121 acgccttcgt gatcgagggc gagggcaagg gcaagcccta cgagggcacc cagaccgcca
     3181 acctgaccgt gaaggagggc gcccccctgc ccttcagcta cgacatcctg accaccgccg
     3241 tgcactacgg caaccgggtg ttcaccaagt accccgagga catccccgac tacttcaagc
     3301 agagcttccc cgagggctac agctgggagc gcaccatgac cttcgaggac aagggcatct
     3361 gcaccatccg cagcgacatc agcctggagg gcgactgctt cttccagaac gtgcgcttca
     3421 agggcaccaa cttccccccc aacggccccg tgatgcagaa gaaaaccctg aagtgggagc
     3481 ccagcaccga gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc aacatggccc
     3541 tgctgctgga gggcggcggc cactacctgt gcgacttcaa gaccacctac aaggccaaga
     3601 aggtggtgca gctgcccgac gcccacttcg tggaccaccg catcgagatc ctgggcaacg
     3661 acagcgacta caacaaggtg aagctgtacg agcacgccgt ggcccgctac agccccctgc
     3721 ccagccaggt gtggtaatct agatcataat cagccatacc acatttgtag aggttttact
     3781 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     3841 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3901 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3961 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     4021 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg cggatcctgc ggccgcaagg
     4081 gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa taaaattgta
     4141 aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta aaagtattga
     4201 tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag tacagtaatc
     4261 aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct tcagcaagcc
     4321 cagcagatcc actagatctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     4381 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4441 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4501 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4561 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4621 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4681 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4741 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4801 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4861 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4921 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4981 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5041 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5101 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5161 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5221 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5281 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5341 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5401 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5461 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5521 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5581 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5641 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5701 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5761 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5821 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5881 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5941 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6001 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6061 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6121 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6181 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6241 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6301 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6361 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6421 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6481 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6541 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6601 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6661 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6721 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6781 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6841 gatttagagc ttgacgggga aagccggcca caatgacggc tactacatgg tgccattcct
     6901 tcctctttat aggaatggag actacctcct gtccaacaat gctcttggat acgagtacgc
     6961 ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc tgcaaaagac
     7021 gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga ggtttgtcca
     7081 ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga aagtacaagt
     7141 acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc ttacttgagt
     7201 aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta aaaggctagc
     7261 aagatctgct cgagatacat ttgattagaa gagtttgact tgggttgatt atagcaaata
     7321 gtggaacaca aacagatttt gaaatattct tgcaaattta tttattaaat ccattcaaat
     7381 attgtcattt tttgtactgt gtaatttata ttgatttgct catacaatat ttacatttcg
     7441 cttaaacaat acactattat ttatatttaa aaatagctat atataaatca aatgtatagc
     7501 tcttttttac acaattaatc aaagctaaaa tatttgtcac acaaatacag taagccactt
     7561 cacaatgata tcttcaaaat ctgtgcttga ctgaatcatt tcatttattg ttttgtaatt
     7621 cacattatac cttacgttct ccaggcacag tttgaattgt tgataaatga agataataca
     7681 tctgatttaa ttagtctcct ttgtttataa taactttaat ctaatttgat agtaataatt
     7741 ccagcaatac atccattaca ataaaaatcg cttttttatt ttgcacatta gtttgccggt
     7801 atttgtttaa agtttcacca tacttttaaa tcatgtgcta tttgaaaagg gctgttatga
     7861 ctggaattaa atgttttttt ttatatcagt gttttagatt gaagtacaat atttataggt
     7921 aaaattcacc agcata
//
