LOCUS       sequence-182572-        7816 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 7816)
  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 7816)
  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 7a-2-Dendra2
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1630..1639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2822..3514
                     /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    3625..3746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3662..3681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3716..3735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3870..4057,4058..4069)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4124..4144)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4124..4142)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4163..4179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4163..4179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4176..4198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4187..4203
                     /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(4211..4217,4218..4235,4236..4241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4256..4277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4394..4411)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4565..5153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4645..4664)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5324..6115,6116..6184))
                     /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     5947..5966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6185..6289)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6452..6473)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6869..6880,6881..7018)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gttattgtca gtttattgca cactgggttg cattaagctg cagttttttc acaacagttt
       61 tccacattgt aacagtcagt ctccaaatta ttccatcagt cctcaaattt agtcatttga
      121 ggccatgaaa acacttaaaa ctacatgaag gtagtttggc attaggtttt agacttgttc
      181 ttgtttggca tattgcaacc tgtgtaaaaa tgatttgatt tttatttttt tttttttttt
      241 ttcagaaaga aacggtccaa aaagagttat gtgaacattt atccatgtgt ttaatgtttt
      301 ttaaattaaa aaaaaacatt tttattaaac cacttgtgac taaacctttt ttagttattc
      361 tattttgtga cgagtctgaa tggccaaagt actcttgaat ccaaaatctt tatagtaaaa
      421 tgtttaatct tacttgcagt gttgggtata atttgtaaca aagttgaatt ccatatctgc
      481 tgagtgaaaa agtaaagtaa gagattgctg taaatattta ggttatttat ttattaatac
      541 aatatagaaa gtaacaaaac acaatgtaaa aaatcaataa agaaagtaaa tattaaaagt
      601 aatgataaac taatgatgta aacaatatta gcaacaaagt ttgtacaatt cagggagggt
      661 aaagaatttc acctaatagt caaactctgt attaaataat ataaaatctt tgttcttagt
      721 ctgaagagga gactgtgaga gaaagcttac tggttgttaa attagaaaat ttgtaacttg
      781 cagattaaaa tgatttaaat aattttataa aggccatgtt tgaaacaaat gtaaaaacat
      841 aataaatgtt aagaatttca gatattgaga ttttacaatt taaaaaaagc aataaagtaa
      901 tagcagagac ctacactctt aaagtaaatt acccaactct gcttactagt aacttttgga
      961 atagttacta gtatccattg aacaaatact agaatctaat taaattgtgt gagtgtccaa
     1021 tgaaaagatt cttgtattta aaacagtacc agtatctata tatgtacaaa atttagtgct
     1081 agcaaaatgc aaataaatac tactgcatat aatggattgc attgtaaatc gactcgccat
     1141 aatacattta tttcctattt ttacttttaa caaaaaataa gttcactgag tatttcatgt
     1201 cattaaatcg cattaaacct cttgcatgct ttatgtacac tagtgcagtt cgctaaatga
     1261 ccaccggtgt cactaataac acgatcgttt ctgaattctg cattaaacgc catttatacg
     1321 ttgctcttat tagtaaacaa tttctccatc atgagctcga ccagtattgc atatttcatc
     1381 ctcttaaatc cagagttgtt tcttttaatc actttctctt catgataaaa gcagtttttg
     1441 gttttcagaa gtttacaaca aacccagtca gttattgcct cagcaaacac tggagcaact
     1501 gcacacttgt gaaaagcttc ttgcgaaaca caaacctttc tctggactta taaataaagg
     1561 cattcagcag ctccaccata cacctccact ggtcttgata aagcagatat cagcagtgat
     1621 acaggtaccg ccaccatgga tgaggaaatc gctgccctgg tcgttgacaa cggctccggt
     1681 atgtgcaaag ccggttttgc tggagatgat gcccctcgtg ctgttttccc ctccattgtt
     1741 ggacgaccca gacatcaggt acatttaata aaccatccta ataacatgtt aatcatattg
     1801 aaccacatta aactctcatt tgtgcatttt acatctttta aaaccgattt atattcgttt
     1861 ttagataatg aagtttatta aagtgttaat tcacagcttt attgcttgtt ttgagaatct
     1921 gcttgcataa gtgattttta aacttgatct gattttcgtt tgtttcgaag ttcttcagct
     1981 ttgaaagtca gatcttcaat tcacagccat gccatagttt taggatacag tataagtaga
     2041 cgaattggga tgcatcgtag attttatgct gtgtttgtta tggtttttca gtggtagaga
     2101 ttttcagagt attattaggg ttatagtcag tgtcaggttt atttatcgtc agcgtgatgg
     2161 gcaaaaaaca gctggcagta cgggcttgac atcaagttgc gtgctgggag gctgcttcca
     2221 catattttgg gcattatgga agactagtga ttttgttgtt gtgacgtcat cagtactgac
     2281 aacaagtcac agtctgcctc agtgaccaga atccactcgt catgcacttt ttcatccagc
     2341 aacaaagatg acattgtgcc tatatgtagt ctgagatgtt aaaaggttta aatatttcta
     2401 taacaatatt ccatcacatt ctgggagcaa gccaactgct gttactctcg cccagaaatc
     2461 tgcgccaaca tggacacacc cagtggagca agccatctga ttttcatggt cagattgtaa
     2521 agaagcaatt actcttggac agtttaagca gaaaagccac caagtcaagt cactggtcat
     2581 atgatgaact cgaagatcta ataaaagtta atttaagcgt tcattacaat aaattcaagt
     2641 gaatctgttt taagctagcc tttaacgttt ttaaattcct tcaaacaaat tagttcctct
     2701 ttgcagttcc tcatttctcg gtgagctggt gctttaatcc ctttactttt tcttcctcta
     2761 gggagtgatg gttggcatgg gacgaattaa ttcctgcagc ccgggggtac cggtcgccac
     2821 catgaacacc ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg tgcacatgga
     2881 gggcaacgtg aacggccacg ccttcgtgat cgagggcgag ggcaagggca agccctacga
     2941 gggcacccag accgccaacc tgaccgtgaa ggagggcgcc cccctgccct tcagctacga
     3001 catcctgacc accgccgtgc actacggcaa ccgggtgttc accaagtacc ccgaggacat
     3061 ccccgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca ccatgacctt
     3121 cgaggacaag ggcatctgca ccatccgcag cgacatcagc ctggagggcg actgcttctt
     3181 ccagaacgtg cgcttcaagg gcaccaactt cccccccaac ggccccgtga tgcagaagaa
     3241 aaccctgaag tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc tgctggtggg
     3301 caacatcaac atggccctgc tgctggaggg cggcggccac tacctgtgcg acttcaagac
     3361 cacctacaag gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg accaccgcat
     3421 cgagatcctg ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc acgccgtggc
     3481 ccgctacagc cccctgccca gccaggtgtg gtaatctaga tcataatcag ccataccaca
     3541 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     3601 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     3661 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3721 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
     3781 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcgg
     3841 atcctgcggc cgcaagggaa aatagaatga agtgatctcc aaaaaataag tactttttga
     3901 ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat gtaattaagt
     3961 aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc caaaaataat
     4021 acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg ttcttgaccc
     4081 cctaccttca gcaagcccag cagatccact agatcttttg ttccctttag tgagggttaa
     4141 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4201 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4261 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4321 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4381 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4441 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4501 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4561 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4621 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4681 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4741 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4801 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4861 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4921 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4981 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5041 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5101 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5161 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5221 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5281 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5341 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5401 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5461 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5521 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5581 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5641 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5701 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5761 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5821 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5881 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5941 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6001 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6061 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6121 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6181 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6241 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6301 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     6361 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6421 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6481 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6541 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6601 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggccacaa tgacggctac
     6661 tacatggtgc cattccttcc tctttatagg aatggagact acctcctgtc caacaatgct
     6721 cttggatacg agtacgccta cctgttggac ccaggtcatt gcacaacacc agaaatgccc
     6781 tctgatctgc aaaagacgtg aatatctgtt cagacaccca tatccactct gttccacaca
     6841 ggtcagaggt ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa aaattttact
     6901 caagtgaaag tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc
     6961 tagaatctta cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa
     7021 ggaagtaaaa ggctagcaag atctgctcga gatacatttg attagaagag tttgacttgg
     7081 gttgattata gcaaatagtg gaacacaaac agattttgaa atattcttgc aaatttattt
     7141 attaaatcca ttcaaatatt gtcatttttt gtactgtgta atttatattg atttgctcat
     7201 acaatattta catttcgctt aaacaataca ctattattta tatttaaaaa tagctatata
     7261 taaatcaaat gtatagctct tttttacaca attaatcaaa gctaaaatat ttgtcacaca
     7321 aatacagtaa gccacttcac aatgatatct tcaaaatctg tgcttgactg aatcatttca
     7381 tttattgttt tgtaattcac attatacctt acgttctcca ggcacagttt gaattgttga
     7441 taaatgaaga taatacatct gatttaatta gtctcctttg tttataataa ctttaatcta
     7501 atttgatagt aataattcca gcaatacatc cattacaata aaaatcgctt ttttattttg
     7561 cacattagtt tgccggtatt tgtttaaagt ttcaccatac ttttaaatca tgtgctattt
     7621 gaaaagggct gttatgactg gaattaaatg ttttttttta tatcagtgtt ttagattgaa
     7681 gtacaatatt tataggtaaa attcaccagc atatgcttaa agatagccat gctccaggtc
     7741 cttgtgtcct tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc
     7801 tccatttttc tgtgaa
//
