LOCUS       sequence-182592-        8023 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 8023)
  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 8023)
  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 99-1-Dendra2
FEATURES             Location/Qualifiers
     source          1..8023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1739..1748
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3138..3830
                     /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    3941..4062
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3978..3997)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4032..4051
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4186..4373,4374..4385)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4440..4460)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4440..4458)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4479..4495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4479..4495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4492..4514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4503..4519
                     /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(4527..4533,4534..4551,4552..4557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4572..4593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4710..4727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4881..5469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4961..4980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5640..6431,6432..6500))
                     /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     6263..6282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6501..6605)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6768..6789)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7185..7196,7197..7334)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 agcatatgct taaagatagc catgctccag gtccttgtgt ccttctaagg tctgtttgtg
       61 gtttaagctg tttgttgaca cacgcttgtc atctccattt ttctgtgaag ttattgtcag
      121 tttattgcac actgggttgc attaagctgc agttttttca caacagtttt ccacattgta
      181 acagtcagtc tccaaattat tccatcagtc ctcaaattta gtcatttgag gccatgaaaa
      241 cacttaaaac tacatgaagg tagtttggca ttaggtttta gacttgttct tgtttggcat
      301 attgcaacct gtgtaaaaat gatttgattt ttattttttt tttttttttt tcagaaagaa
      361 acggtccaaa aagagttatg tgaacattta tccatgtgtt taatgttttt taaattaaaa
      421 aaaaacattt ttattaaacc acttgtgact aaaccttttt tagttattct attttgtgac
      481 gagtctgaat ggccaaagta ctcttgaatc caaaatcttt atagtaaaat gtttaatctt
      541 acttgcagtg ttgggtataa tttgtaacaa agttgaattc catatctgct gagtgaaaaa
      601 gtaaagtaag agattgctgt aaatatttag gttatttatt tattaataca atatagaaag
      661 taacaaaaca caatgtaaaa aatcaataaa gaaagtaaat attaaaagta atgataaact
      721 aatgatgtaa acaatattag caacaaagtt tgtacaattc agggagggta aagaatttca
      781 cctaatagtc aaactctgta ttaaataata taaaatcttt gttcttagtc tgaagaggag
      841 actgtgagag aaagcttact ggttgttaaa ttagaaaatt tgtaacttgc agattaaaat
      901 gatttaaata attttataaa ggccatgttt gaaacaaatg taaaaacata ataaatgtta
      961 agaatttcag atattgagat tttacaattt aaaaaaagca ataaagtaat agcagagacc
     1021 tacactctta aagtaaatta cccaactctg cttactagta acttttggaa tagttactag
     1081 tatccattga acaaatacta gaatctaatt aaattgtgtg agtgtccaat gaaaagattc
     1141 ttgtatttaa aacagtacca gtatctatat atgtacaaaa tttagtgcta gcaaaatgca
     1201 aataaatact actgcatata atggattgca ttgtaaatcg actcgccata atacatttat
     1261 ttcctatttt tacttttaac aaaaaataag ttcactgagt atttcatgtc attaaatcgc
     1321 attaaacctc ttgcatgctt tatgtacact agtgcagttc gctaaatgac caccggtgtc
     1381 actaataaca cgatcgtttc tgaattctgc attaaacgcc atttatacgt tgctcttatt
     1441 agtaaacaat ttctccatca tgagctcgac cagtattgca tatttcatcc tcttaaatcc
     1501 agagttgttt cttttaatca ctttctcttc atgataaaag cagtttttgg ttttcagaag
     1561 tttacaacaa acccagtcag ttattgcctc agcaaacact ggagcaactg cacacttgtg
     1621 aaaagcttct tgcgaaacac aaacctttct ctggacttat aaataaaggc attcagcagc
     1681 tccaccatac acctccactg gtcttgataa agcagatatc agcagtgata caggtaccgc
     1741 caccatggat gaggaaatcg ctgccctggt cgttgacaac ggctccggta tgtgcaaagc
     1801 cggttttgct ggagatgatg cccctcgtgc tgttttcccc tccattgttg gacgacccag
     1861 acatcaggta catttaataa accatcctaa taacatgtta atcatattga accacattaa
     1921 actctcattt gtgcatttta catcttttaa aaccgattta tattcgtttt tagataatga
     1981 agtttattaa agtgttaatt cacagcttta ttgcttgttt tgagaatctg cttgcataag
     2041 tgatttttaa acttgatctg attttcgttt gtttcgaagt tcttcagctt tgaaagtcag
     2101 atcttcaatt cacagccatg ccatagtttt aggatacagt ataagtagac gaattgggat
     2161 gcatcgtaga ttttatgctg tgtttgttat ggtttttcag tggtagagat tttcagagta
     2221 ttattagggt tatagtcagt gtcaggttta tttatcgtca gcgtgatggg caaaaaacag
     2281 ctggcagtac gggctacgga tagctatgcc aatgcacatt tctctatctg caacaagcct
     2341 aattaaaaga aagaatgcaa tgcacatctt tgtgaaggtc atacgctgat gtaccagaag
     2401 atattatcat cagtcattat gtctctagag caggaaaatc cctgcctttt ttcaagtcca
     2461 tgatcaaatg agcagccact tgtcattaac ccgtagatcc gatcttgtga taagtttgat
     2521 ggcacaagct cgattctatg ggtctctgtc tctgtggtag ataatcgacc aacatgtaga
     2581 gccagcctgc tttttttttt attttattat ttttttccct ttgcttgtca ggcactgaag
     2641 ttgctcagac ttgtccacag taattttaca atcgattgac atttatgtaa acttcttcaa
     2701 aagggattac agcaactgag agtgtgtgca tccaggctga ggtagagcca actgctgtta
     2761 ctctcgccca gaaatctgcg ccaacatgga cacacccagt ggagcaagcc atctgatttt
     2821 catggtcaga ttgtaaagaa gcaattactc ttggacagtt taagcagaaa agccaccaag
     2881 tcaagtcact ggtcatatga tgaactcgaa gatctaataa aagttaattt aagcgttcat
     2941 tacaataaat tcaagtgaat ctgttttaag ctagccttta acgtttttaa attccttcaa
     3001 acaaattagt tcctctttgc agttcctcat ttctcggtga gctggtgctt taatcccttt
     3061 actttttctt cctctaggga gtgatggttg gcatgggacg aattaattcc tgcagcccgg
     3121 gggtaccggt cgccaccatg aacaccccgg gaattaacct gatcaaggag gacatgcgcg
     3181 tgaaggtgca catggagggc aacgtgaacg gccacgcctt cgtgatcgag ggcgagggca
     3241 agggcaagcc ctacgagggc acccagaccg ccaacctgac cgtgaaggag ggcgcccccc
     3301 tgcccttcag ctacgacatc ctgaccaccg ccgtgcacta cggcaaccgg gtgttcacca
     3361 agtaccccga ggacatcccc gactacttca agcagagctt ccccgagggc tacagctggg
     3421 agcgcaccat gaccttcgag gacaagggca tctgcaccat ccgcagcgac atcagcctgg
     3481 agggcgactg cttcttccag aacgtgcgct tcaagggcac caacttcccc cccaacggcc
     3541 ccgtgatgca gaagaaaacc ctgaagtggg agcccagcac cgagaagctg cacgtgcgcg
     3601 acggcctgct ggtgggcaac atcaacatgg ccctgctgct ggagggcggc ggccactacc
     3661 tgtgcgactt caagaccacc tacaaggcca agaaggtggt gcagctgccc gacgcccact
     3721 tcgtggacca ccgcatcgag atcctgggca acgacagcga ctacaacaag gtgaagctgt
     3781 acgagcacgc cgtggcccgc tacagccccc tgcccagcca ggtgtggtaa tctagatcat
     3841 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3901 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3961 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4021 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     4081 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4141 aggccgaaat cggcggatcc tgcggccgca agggaaaata gaatgaagtg atctccaaaa
     4201 aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa
     4261 aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa
     4321 aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag tactttacac
     4381 ctctggttct tgacccccta ccttcagcaa gcccagcaga tccactagat cttttgttcc
     4441 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     4501 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4561 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4681 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4741 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4801 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4861 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4921 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4981 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5041 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5101 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5161 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5221 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5281 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5341 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5401 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5461 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5521 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5581 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5641 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5701 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5761 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5821 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5881 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5941 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6001 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6061 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6121 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6181 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6241 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6301 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6361 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6421 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6481 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6541 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6601 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     6661 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     6721 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     6781 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     6841 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     6901 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     6961 ccacaatgac ggctactaca tggtgccatt ccttcctctt tataggaatg gagactacct
     7021 cctgtccaac aatgctcttg gatacgagta cgcctacctg ttggacccag gtcattgcac
     7081 aacaccagaa atgccctctg atctgcaaaa gacgtgaata tctgttcaga cacccatatc
     7141 cactctgttc cacacaggtc agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt
     7201 actcaaaaat tttactcaag tgaaagtaca agtacttagg gaaaatttta ctcaattaaa
     7261 agtaaaagta tctggctaga atcttacttg agtaaaagta aaaaagtact ccattaaaat
     7321 tgtacttgag tattaaggaa gtaaaaggct agcaagatct gctcgagata catttgatta
     7381 gaagagtttg acttgggttg attatagcaa atagtggaac acaaacagat tttgaaatat
     7441 tcttgcaaat ttatttatta aatccattca aatattgtca ttttttgtac tgtgtaattt
     7501 atattgattt gctcatacaa tatttacatt tcgcttaaac aatacactat tatttatatt
     7561 taaaaatagc tatatataaa tcaaatgtat agctcttttt tacacaatta atcaaagcta
     7621 aaatatttgt cacacaaata cagtaagcca cttcacaatg atatcttcaa aatctgtgct
     7681 tgactgaatc atttcattta ttgttttgta attcacatta taccttacgt tctccaggca
     7741 cagtttgaat tgttgataaa tgaagataat acatctgatt taattagtct cctttgttta
     7801 taataacttt aatctaattt gatagtaata attccagcaa tacatccatt acaataaaaa
     7861 tcgctttttt attttgcaca ttagtttgcc ggtatttgtt taaagtttca ccatactttt
     7921 aaatcatgtg ctatttgaaa agggctgtta tgactggaat taaatgtttt tttttatatc
     7981 agtgttttag attgaagtac aatatttata ggtaaaattc acc
//
