LOCUS       sequence-182663-        8353 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 8353)
  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 8353)
  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 214-Dendra2
FEATURES             Location/Qualifiers
     source          1..8353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1657..1666
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3386..4078
                     /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    4189..4310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4226..4245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4280..4299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4434..4621,4622..4633)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4688..4708)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4688..4706)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4727..4743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4727..4743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4740..4762)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4751..4767
                     /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(4775..4781,4782..4799,4800..4805))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4820..4841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4958..4975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5129..5717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5209..5228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5888..6679,6680..6748))
                     /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     6511..6530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6749..6853)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7016..7037)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7433..7444,7445..7582)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 cgcttgtcat ctccattttt ctgtgaagtt attgtcagtt tattgcacac tgggttgcat
       61 taagctgcag ttttttcaca acagttttcc acattgtaac agtcagtctc caaattattc
      121 catcagtcct caaatttagt catttgaggc catgaaaaca cttaaaacta catgaaggta
      181 gtttggcatt aggttttaga cttgttcttg tttggcatat tgcaacctgt gtaaaaatga
      241 tttgattttt attttttttt tttttttttc agaaagaaac ggtccaaaaa gagttatgtg
      301 aacatttatc catgtgttta atgtttttta aattaaaaaa aaacattttt attaaaccac
      361 ttgtgactaa acctttttta gttattctat tttgtgacga gtctgaatgg ccaaagtact
      421 cttgaatcca aaatctttat agtaaaatgt ttaatcttac ttgcagtgtt gggtataatt
      481 tgtaacaaag ttgaattcca tatctgctga gtgaaaaagt aaagtaagag attgctgtaa
      541 atatttaggt tatttattta ttaatacaat atagaaagta acaaaacaca atgtaaaaaa
      601 tcaataaaga aagtaaatat taaaagtaat gataaactaa tgatgtaaac aatattagca
      661 acaaagtttg tacaattcag ggagggtaaa gaatttcacc taatagtcaa actctgtatt
      721 aaataatata aaatctttgt tcttagtctg aagaggagac tgtgagagaa agcttactgg
      781 ttgttaaatt agaaaatttg taacttgcag attaaaatga tttaaataat tttataaagg
      841 ccatgtttga aacaaatgta aaaacataat aaatgttaag aatttcagat attgagattt
      901 tacaatttaa aaaaagcaat aaagtaatag cagagaccta cactcttaaa gtaaattacc
      961 caactctgct tactagtaac ttttggaata gttactagta tccattgaac aaatactaga
     1021 atctaattaa attgtgtgag tgtccaatga aaagattctt gtatttaaaa cagtaccagt
     1081 atctatatat gtacaaaatt tagtgctagc aaaatgcaaa taaatactac tgcatataat
     1141 ggattgcatt gtaaatcgac tcgccataat acatttattt cctattttta cttttaacaa
     1201 aaaataagtt cactgagtat ttcatgtcat taaatcgcat taaacctctt gcatgcttta
     1261 tgtacactag tgcagttcgc taaatgacca ccggtgtcac taataacacg atcgtttctg
     1321 aattctgcat taaacgccat ttatacgttg ctcttattag taaacaattt ctccatcatg
     1381 agctcgacca gtattgcata tttcatcctc ttaaatccag agttgtttct tttaatcact
     1441 ttctcttcat gataaaagca gtttttggtt ttcagaagtt tacaacaaac ccagtcagtt
     1501 attgcctcag caaacactgg agcaactgca cacttgtgaa aagcttcttg cgaaacacaa
     1561 acctttctct ggacttataa ataaaggcat tcagcagctc caccatacac ctccactggt
     1621 cttgataaag cagatatcag cagtgataca ggtaccgcca ccatggatga ggaaatcgct
     1681 gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg agatgatgcc
     1741 cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca tttaataaac
     1801 catcctaata acatgttaat catattgaac cacattaaac tctcatttgt gcattttaca
     1861 tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag tgttaattca
     1921 cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac ttgatctgat
     1981 tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca cagccatgcc
     2041 atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt ttatgctgtg
     2101 tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta tagtcagtgt
     2161 caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg gctaatggaa
     2221 gcagagagtc ggtgggggta tactaagaaa agggagcaag caaaatagac aggaaaaaaa
     2281 gccgggggga aagatggatt cgtcaaaact tgaaatcaac gagtggaagt ttgatttttg
     2341 ggttactgca tttgaatatt gcactgtaaa aatgaccaca acaaatgttt ttgtcacttt
     2401 catttggtat caggtaaact atttttgtat ttttacaaag ttaaacttaa tctttttagt
     2461 tactttgact aaagttgaga tgacttaaaa agtttagttt attcaactaa ataacaatat
     2521 ttttacagtc tacacttaat tgtaatttat taaactatgt taatcgtgtt ccaacttaat
     2581 ttgatgagtt atgcaagtca gttcaatata aaaaataagt tcaaattact caaaaggttc
     2641 atttgattta aattttaagg caaccagtat tttcttttac agtgtactgc cttggttaac
     2701 aatatcaaaa ttctgacgtg tattaaatgt aatgatgtga atacaaatgt ttataaccaa
     2761 tgtttcaata ttctagtaac attatcttta tcctctctct tgctctttgc ttccggcttc
     2821 cggtcgggag aagcaggctg tgctgccatt gactgagagc gttgtctgtc tgcctgtcta
     2881 cacttgctgt gcagaacttc ctgcacctgt acagcaggca cagacaggca gacagatggc
     2941 agcccgcctg acaggccaca cagtgtgaga ccagcccttg agttcctgca aacagccaac
     3001 tgctgttact ctcgcccaga aatctgcgcc aacatggaca cacccagtgg agcaagccat
     3061 ctgattttca tggtcagatt gtaaagaagc aattactctt ggacagttta agcagaaaag
     3121 ccaccaagtc aagtcactgg tcatatgatg aactcgaaga tctaataaaa gttaatttaa
     3181 gcgttcatta caataaattc aagtgaatct gttttaagct agcctttaac gtttttaaat
     3241 tccttcaaac aaattagttc ctctttgcag ttcctcattt ctcggtgagc tggtgcttta
     3301 atccctttac tttttcttcc tctagggagt gatggttggc atgggacgaa ttaattcctg
     3361 cagcccgggg gtaccggtcg ccaccatgaa caccccggga attaacctga tcaaggagga
     3421 catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc cacgccttcg tgatcgaggg
     3481 cgagggcaag ggcaagccct acgagggcac ccagaccgcc aacctgaccg tgaaggaggg
     3541 cgcccccctg cccttcagct acgacatcct gaccaccgcc gtgcactacg gcaaccgggt
     3601 gttcaccaag taccccgagg acatccccga ctacttcaag cagagcttcc ccgagggcta
     3661 cagctgggag cgcaccatga ccttcgagga caagggcatc tgcaccatcc gcagcgacat
     3721 cagcctggag ggcgactgct tcttccagaa cgtgcgcttc aagggcacca acttcccccc
     3781 caacggcccc gtgatgcaga agaaaaccct gaagtgggag cccagcaccg agaagctgca
     3841 cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc ctgctgctgg agggcggcgg
     3901 ccactacctg tgcgacttca agaccaccta caaggccaag aaggtggtgc agctgcccga
     3961 cgcccacttc gtggaccacc gcatcgagat cctgggcaac gacagcgact acaacaaggt
     4021 gaagctgtac gagcacgccg tggcccgcta cagccccctg cccagccagg tgtggtaatc
     4081 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     4141 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     4201 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4261 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     4321 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4381 taaccaatag gccgaaatcg gcggatcctg cggccgcaag ggaaaataga atgaagtgat
     4441 ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt
     4501 ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt
     4561 aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta
     4621 ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc cactagatct
     4681 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     4741 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4801 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4861 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4921 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4981 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5041 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5101 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5161 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5221 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5281 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5341 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5401 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5461 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5521 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5581 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5641 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5701 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5761 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5821 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5881 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5941 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6001 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6061 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6121 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6181 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6241 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6301 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6361 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6421 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6481 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6541 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6601 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6661 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6721 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6781 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6841 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6901 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6961 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     7021 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     7081 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     7141 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     7201 aaagccggcc acaatgacgg ctactacatg gtgccattcc ttcctcttta taggaatgga
     7261 gactacctcc tgtccaacaa tgctcttgga tacgagtacg cctacctgtt ggacccaggt
     7321 cattgcacaa caccagaaat gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca
     7381 cccatatcca ctctgttcca cacaggtcag aggtttgtcc aggagttctt gacagaggtg
     7441 taaaaagtac tcaaaaattt tactcaagtg aaagtacaag tacttaggga aaattttact
     7501 caattaaaag taaaagtatc tggctagaat cttacttgag taaaagtaaa aaagtactcc
     7561 attaaaattg tacttgagta ttaaggaagt aaaaggctag caagatctgc tcgagataca
     7621 tttgattaga agagtttgac ttgggttgat tatagcaaat agtggaacac aaacagattt
     7681 tgaaatattc ttgcaaattt atttattaaa tccattcaaa tattgtcatt ttttgtactg
     7741 tgtaatttat attgatttgc tcatacaata tttacatttc gcttaaacaa tacactatta
     7801 tttatattta aaaatagcta tatataaatc aaatgtatag ctctttttta cacaattaat
     7861 caaagctaaa atatttgtca cacaaataca gtaagccact tcacaatgat atcttcaaaa
     7921 tctgtgcttg actgaatcat ttcatttatt gttttgtaat tcacattata ccttacgttc
     7981 tccaggcaca gtttgaattg ttgataaatg aagataatac atctgattta attagtctcc
     8041 tttgtttata ataactttaa tctaatttga tagtaataat tccagcaata catccattac
     8101 aataaaaatc gcttttttat tttgcacatt agtttgccgg tatttgttta aagtttcacc
     8161 atacttttaa atcatgtgct atttgaaaag ggctgttatg actggaatta aatgtttttt
     8221 tttatatcag tgttttagat tgaagtacaa tatttatagg taaaattcac cagcatatgc
     8281 ttaaagatag ccatgctcca ggtccttgtg tccttctaag gtctgtttgt ggtttaagct
     8341 gtttgttgac aca
//
