LOCUS       sequence-182644-        7886 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 7886)
  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 7886)
  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 187-dendra2
FEATURES             Location/Qualifiers
     source          1..7886
                     /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             2892..3584
                     /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    3695..3816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3732..3751)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3786..3805
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3940..4127,4128..4139)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4194..4214)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4194..4212)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4233..4249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4233..4249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4246..4268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4257..4273
                     /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(4281..4287,4288..4305,4306..4311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4326..4347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4464..4481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4635..5223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4715..4734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5394..6185,6186..6254))
                     /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     6017..6036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6255..6359)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6522..6543)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6939..6950,6951..7088)
                     /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 cgggctctcc gcagttcagc agcagcagaa gaggcagaga
     2221 tgaaggtgac ctgtggctgg gccaggggct gcaacacagg acatgggagc tgtctctcac
     2281 tcccgctcgt gtcttgtgtt gcagccagtg gaacggctac actgtccaca tctgagaccg
     2341 actgagaaca ttacacccat tatcttctgc tgagaggaaa aaacagggct gatgatgatg
     2401 cgtaagtttc tgaataaaac gcattcacag tttatttgat aagtgcaaag caaaggtttt
     2461 aggaattaaa attcagatta tacgtagttg aaagcaccta gccaactgct gttactctcg
     2521 cccagaaatc tgcgccaaca tggacacacc cagtggagca agccatctga ttttcatggt
     2581 cagattgtaa agaagcaatt actcttggac agtttaagca gaaaagccac caagtcaagt
     2641 cactggtcat atgatgaact cgaagatcta ataaaagtta atttaagcgt tcattacaat
     2701 aaattcaagt gaatctgttt taagctagcc tttaacgttt ttaaattcct tcaaacaaat
     2761 tagttcctct ttgcagttcc tcatttctcg gtgagctggt gctttaatcc ctttactttt
     2821 tcttcctcta gggagtgatg gttggcatgg gacgaattaa ttcctgcagc ccgggggtac
     2881 cggtcgccac catgaacacc ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg
     2941 tgcacatgga gggcaacgtg aacggccacg ccttcgtgat cgagggcgag ggcaagggca
     3001 agccctacga gggcacccag accgccaacc tgaccgtgaa ggagggcgcc cccctgccct
     3061 tcagctacga catcctgacc accgccgtgc actacggcaa ccgggtgttc accaagtacc
     3121 ccgaggacat ccccgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca
     3181 ccatgacctt cgaggacaag ggcatctgca ccatccgcag cgacatcagc ctggagggcg
     3241 actgcttctt ccagaacgtg cgcttcaagg gcaccaactt cccccccaac ggccccgtga
     3301 tgcagaagaa aaccctgaag tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc
     3361 tgctggtggg caacatcaac atggccctgc tgctggaggg cggcggccac tacctgtgcg
     3421 acttcaagac cacctacaag gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg
     3481 accaccgcat cgagatcctg ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc
     3541 acgccgtggc ccgctacagc cccctgccca gccaggtgtg gtaatctaga tcataatcag
     3601 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3661 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     3721 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3781 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     3841 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3901 aaatcggcgg atcctgcggc cgcaagggaa aatagaatga agtgatctcc aaaaaataag
     3961 tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat
     4021 gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc
     4081 caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg
     4141 ttcttgaccc cctaccttca gcaagcccag cagatccact agatcttttg ttccctttag
     4201 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4261 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4321 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4381 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4441 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4501 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4561 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4621 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4681 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4741 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4801 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4861 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4921 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4981 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5041 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5101 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5161 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5221 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5281 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5341 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5401 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5461 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5521 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5581 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5641 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5701 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5761 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5821 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5881 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5941 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6001 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6061 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6361 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6421 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6481 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6541 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6601 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6661 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggccacaa
     6721 tgacggctac tacatggtgc cattccttcc tctttatagg aatggagact acctcctgtc
     6781 caacaatgct cttggatacg agtacgccta cctgttggac ccaggtcatt gcacaacacc
     6841 agaaatgccc tctgatctgc aaaagacgtg aatatctgtt cagacaccca tatccactct
     6901 gttccacaca ggtcagaggt ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa
     6961 aaattttact caagtgaaag tacaagtact tagggaaaat tttactcaat taaaagtaaa
     7021 agtatctggc tagaatctta cttgagtaaa agtaaaaaag tactccatta aaattgtact
     7081 tgagtattaa ggaagtaaaa ggctagcaag atctgctcga gatacatttg attagaagag
     7141 tttgacttgg gttgattata gcaaatagtg gaacacaaac agattttgaa atattcttgc
     7201 aaatttattt attaaatcca ttcaaatatt gtcatttttt gtactgtgta atttatattg
     7261 atttgctcat acaatattta catttcgctt aaacaataca ctattattta tatttaaaaa
     7321 tagctatata taaatcaaat gtatagctct tttttacaca attaatcaaa gctaaaatat
     7381 ttgtcacaca aatacagtaa gccacttcac aatgatatct tcaaaatctg tgcttgactg
     7441 aatcatttca tttattgttt tgtaattcac attatacctt acgttctcca ggcacagttt
     7501 gaattgttga taaatgaaga taatacatct gatttaatta gtctcctttg tttataataa
     7561 ctttaatcta atttgatagt aataattcca gcaatacatc cattacaata aaaatcgctt
     7621 ttttattttg cacattagtt tgccggtatt tgtttaaagt ttcaccatac ttttaaatca
     7681 tgtgctattt gaaaagggct gttatgactg gaattaaatg ttttttttta tatcagtgtt
     7741 ttagattgaa gtacaatatt tataggtaaa attcaccagc atatgcttaa agatagccat
     7801 gctccaggtc cttgtgtcct tctaaggtct gtttgtggtt taagctgttt gttgacacac
     7861 gcttgtcatc tccatttttc tgtgaa
//
