LOCUS       sequence-182774-        8232 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 8232)
  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 8232)
  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 338-2-Dendra2
FEATURES             Location/Qualifiers
     source          1..8232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1715..1724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3323..4015
                     /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    4126..4247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4163..4182)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4217..4236
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4371..4558,4559..4570)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4625..4645)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4625..4643)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4664..4680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4664..4680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4677..4699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4688..4704
                     /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(4712..4718,4719..4736,4737..4742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4895..4912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5066..5654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5146..5165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5825..6616,6617..6685))
                     /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     6448..6467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6686..6790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6953..6974)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7370..7381,7382..7519)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ctccaggtcc ttgtgtcctt ctaaggtctg tttgtggttt aagctgtttg ttgacacacg
       61 cttgtcatct ccatttttct gtgaagttat tgtcagttta ttgcacactg ggttgcatta
      121 agctgcagtt ttttcacaac agttttccac attgtaacag tcagtctcca aattattcca
      181 tcagtcctca aatttagtca tttgaggcca tgaaaacact taaaactaca tgaaggtagt
      241 ttggcattag gttttagact tgttcttgtt tggcatattg caacctgtgt aaaaatgatt
      301 tgatttttat tttttttttt tttttttcag aaagaaacgg tccaaaaaga gttatgtgaa
      361 catttatcca tgtgtttaat gttttttaaa ttaaaaaaaa acatttttat taaaccactt
      421 gtgactaaac cttttttagt tattctattt tgtgacgagt ctgaatggcc aaagtactct
      481 tgaatccaaa atctttatag taaaatgttt aatcttactt gcagtgttgg gtataatttg
      541 taacaaagtt gaattccata tctgctgagt gaaaaagtaa agtaagagat tgctgtaaat
      601 atttaggtta tttatttatt aatacaatat agaaagtaac aaaacacaat gtaaaaaatc
      661 aataaagaaa gtaaatatta aaagtaatga taaactaatg atgtaaacaa tattagcaac
      721 aaagtttgta caattcaggg agggtaaaga atttcaccta atagtcaaac tctgtattaa
      781 ataatataaa atctttgttc ttagtctgaa gaggagactg tgagagaaag cttactggtt
      841 gttaaattag aaaatttgta acttgcagat taaaatgatt taaataattt tataaaggcc
      901 atgtttgaaa caaatgtaaa aacataataa atgttaagaa tttcagatat tgagatttta
      961 caatttaaaa aaagcaataa agtaatagca gagacctaca ctcttaaagt aaattaccca
     1021 actctgctta ctagtaactt ttggaatagt tactagtatc cattgaacaa atactagaat
     1081 ctaattaaat tgtgtgagtg tccaatgaaa agattcttgt atttaaaaca gtaccagtat
     1141 ctatatatgt acaaaattta gtgctagcaa aatgcaaata aatactactg catataatgg
     1201 attgcattgt aaatcgactc gccataatac atttatttcc tatttttact tttaacaaaa
     1261 aataagttca ctgagtattt catgtcatta aatcgcatta aacctcttgc atgctttatg
     1321 tacactagtg cagttcgcta aatgaccacc ggtgtcacta ataacacgat cgtttctgaa
     1381 ttctgcatta aacgccattt atacgttgct cttattagta aacaatttct ccatcatgag
     1441 ctcgaccagt attgcatatt tcatcctctt aaatccagag ttgtttcttt taatcacttt
     1501 ctcttcatga taaaagcagt ttttggtttt cagaagttta caacaaaccc agtcagttat
     1561 tgcctcagca aacactggag caactgcaca cttgtgaaaa gcttcttgcg aaacacaaac
     1621 ctttctctgg acttataaat aaaggcattc agcagctcca ccatacacct ccactggtct
     1681 tgataaagca gatatcagca gtgatacagg taccgccacc atggatgagg aaatcgctgc
     1741 cctggtcgtt gacaacggct ccggtatgtg caaagccggt tttgctggag atgatgcccc
     1801 tcgtgctgtt ttcccctcca ttgttggacg acccagacat caggtacatt taataaacca
     1861 tcctaataac atgttaatca tattgaacca cattaaactc tcatttgtgc attttacatc
     1921 ttttaaaacc gatttatatt cgtttttaga taatgaagtt tattaaagtg ttaattcaca
     1981 gctttattgc ttgttttgag aatctgcttg cataagtgat ttttaaactt gatctgattt
     2041 tcgtttgttt cgaagttctt cagctttgaa agtcagatct tcaattcaca gccatgccat
     2101 agttttagga tacagtataa gtagacgaat tgggatgcat cgtagatttt atgctgtgtt
     2161 tgttatggtt tttcagtggt agagattttc agagtattat tagggttata gtcagtgtca
     2221 ggtttattta tcgtcagcgt gatgggcaaa aaacagctgg cagtacgggc tcggccttct
     2281 cactctcgtt ttaatagctg ttgggtcttc agtttcagaa gcttggaggt aaactatgat
     2341 gaagtgtatg aaattctcca tgaagaaacg tgaagctccg tgtacctact tggaatgtgt
     2401 gtgatggaag caccagctgc atgttcgctc ctccccacgc ttggcttgat gggactgtgt
     2461 gtgtgtgtgt gtgtgtgtgt gtttctggtg cctgctgaga acaatatcct gatgctgaat
     2521 gagtgtgttg aaggaaactc cagcatcagt gattttgttg ccagaggagc actttgggca
     2581 tcctgtgtat atgtgtgtgt gtttattcag agattgagat tctctacttt taaagcacag
     2641 acaattaaaa aagattaaag gtgccatata ataaaaatct gaatatacct aggtatacag
     2701 tgcatcagga aagtattcat agcgcttcac tttttccaca ttttttattt tacagtctta
     2761 ttccaaaata aattatatta atttattttc tcaaaattct acacacaaca ccccataatg
     2821 acaatgtgaa aaagattttt tttaattgtt gcaaatttat ttgcaacaat tgcactgtag
     2881 ctgaataata agagttcagt atatggccat accatgagcc tcaaacgcaa agccaactgc
     2941 tgttactctc gcccagaaat ctgcgccaac atggacacac ccagtggagc aagccatctg
     3001 attttcatgg tcagattgta aagaagcaat tactcttgga cagtttaagc agaaaagcca
     3061 ccaagtcaag tcactggtca tatgatgaac tcgaagatct aataaaagtt aatttaagcg
     3121 ttcattacaa taaattcaag tgaatctgtt ttaagctagc ctttaacgtt tttaaattcc
     3181 ttcaaacaaa ttagttcctc tttgcagttc ctcatttctc ggtgagctgg tgctttaatc
     3241 cctttacttt ttcttcctct agggagtgat ggttggcatg ggacgaatta attcctgcag
     3301 cccgggggta ccggtcgcca ccatgaacac cccgggaatt aacctgatca aggaggacat
     3361 gcgcgtgaag gtgcacatgg agggcaacgt gaacggccac gccttcgtga tcgagggcga
     3421 gggcaagggc aagccctacg agggcaccca gaccgccaac ctgaccgtga aggagggcgc
     3481 ccccctgccc ttcagctacg acatcctgac caccgccgtg cactacggca accgggtgtt
     3541 caccaagtac cccgaggaca tccccgacta cttcaagcag agcttccccg agggctacag
     3601 ctgggagcgc accatgacct tcgaggacaa gggcatctgc accatccgca gcgacatcag
     3661 cctggagggc gactgcttct tccagaacgt gcgcttcaag ggcaccaact tcccccccaa
     3721 cggccccgtg atgcagaaga aaaccctgaa gtgggagccc agcaccgaga agctgcacgt
     3781 gcgcgacggc ctgctggtgg gcaacatcaa catggccctg ctgctggagg gcggcggcca
     3841 ctacctgtgc gacttcaaga ccacctacaa ggccaagaag gtggtgcagc tgcccgacgc
     3901 ccacttcgtg gaccaccgca tcgagatcct gggcaacgac agcgactaca acaaggtgaa
     3961 gctgtacgag cacgccgtgg cccgctacag ccccctgccc agccaggtgt ggtaatctag
     4021 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     4081 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     4141 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4201 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt
     4261 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4321 ccaataggcc gaaatcggcg gatcctgcgg ccgcaaggga aaatagaatg aagtgatctc
     4381 caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt
     4441 tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa
     4501 gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt
     4561 tacacctctg gttcttgacc ccctaccttc agcaagccca gcagatccac tagatctttt
     4621 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     4681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6781 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     6841 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6901 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6961 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     7021 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     7081 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     7141 gccggccaca atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac
     7201 tacctcctgt ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat
     7261 tgcacaacac cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc
     7321 atatccactc tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa
     7381 aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa
     7441 ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt
     7501 aaaattgtac ttgagtatta aggaagtaaa aggctagcaa gatctgctcg agatacattt
     7561 gattagaaga gtttgacttg ggttgattat agcaaatagt ggaacacaaa cagattttga
     7621 aatattcttg caaatttatt tattaaatcc attcaaatat tgtcattttt tgtactgtgt
     7681 aatttatatt gatttgctca tacaatattt acatttcgct taaacaatac actattattt
     7741 atatttaaaa atagctatat ataaatcaaa tgtatagctc ttttttacac aattaatcaa
     7801 agctaaaata tttgtcacac aaatacagta agccacttca caatgatatc ttcaaaatct
     7861 gtgcttgact gaatcatttc atttattgtt ttgtaattca cattatacct tacgttctcc
     7921 aggcacagtt tgaattgttg ataaatgaag ataatacatc tgatttaatt agtctccttt
     7981 gtttataata actttaatct aatttgatag taataattcc agcaatacat ccattacaat
     8041 aaaaatcgct tttttatttt gcacattagt ttgccggtat ttgtttaaag tttcaccata
     8101 cttttaaatc atgtgctatt tgaaaagggc tgttatgact ggaattaaat gttttttttt
     8161 atatcagtgt tttagattga agtacaatat ttataggtaa aattcaccag catatgctta
     8221 aagatagcca tg
//
