LOCUS       sequence-182658-        7778 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 7778)
  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 7778)
  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 203b-Dendra2
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1766..1775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2920..3612
                     /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    3723..3844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3760..3779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3814..3833
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3968..4155,4156..4167)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4222..4242)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4222..4240)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4261..4277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4261..4277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4274..4296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4285..4301
                     /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(4309..4315,4316..4333,4334..4339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4354..4375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4492..4509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4663..5251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4743..4762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5422..6213,6214..6282))
                     /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     6045..6064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6283..6387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6550..6571)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6967..6978,6979..7116)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gtacaatatt tataggtaaa attcaccagc atatgcttaa agatagccat gctccaggtc
       61 cttgtgtcct tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc
      121 tccatttttc tgtgaagtta ttgtcagttt attgcacact gggttgcatt aagctgcagt
      181 tttttcacaa cagttttcca cattgtaaca gtcagtctcc aaattattcc atcagtcctc
      241 aaatttagtc atttgaggcc atgaaaacac ttaaaactac atgaaggtag tttggcatta
      301 ggttttagac ttgttcttgt ttggcatatt gcaacctgtg taaaaatgat ttgattttta
      361 tttttttttt ttttttttca gaaagaaacg gtccaaaaag agttatgtga acatttatcc
      421 atgtgtttaa tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa
      481 ccttttttag ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa
      541 aatctttata gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt
      601 tgaattccat atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt
      661 atttatttat taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa
      721 agtaaatatt aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt
      781 acaattcagg gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa
      841 aatctttgtt cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta
      901 gaaaatttgt aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa
      961 acaaatgtaa aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa
     1021 aaaagcaata aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt
     1081 actagtaact tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa
     1141 ttgtgtgagt gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg
     1201 tacaaaattt agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg
     1261 taaatcgact cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc
     1321 actgagtatt tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt
     1381 gcagttcgct aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt
     1441 aaacgccatt tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag
     1501 tattgcatat ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg
     1561 ataaaagcag tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc
     1621 aaacactgga gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg
     1681 gacttataaa taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc
     1741 agatatcagc agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt
     1801 tgacaacggc tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt
     1861 tttcccctcc attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa
     1921 catgttaatc atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac
     1981 cgatttatat tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg
     2041 cttgttttga gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt
     2101 tcgaagttct tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg
     2161 atacagtata agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt
     2221 ttttcagtgg tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt
     2281 atcgtcagcg tgatgggcaa aaaacagctg gcagtacggg ctaagcgact catctcaggc
     2341 ttgacatgtg tgttcctcca ggcaggtacc gagcgaggat cccggatcgc tgctgttctc
     2401 cacttaacat gtggcttcct ctttggccga gtggttctca acagttcaac agttcttttg
     2461 atgattgtga aatgttcagg accacttgat cagacgaagg acaatttcac ctctgaaacg
     2521 ggtttgtagc caactgctgt tactctcgcc cagaaatctg cgccaacatg gacacaccca
     2581 gtggagcaag ccatctgatt ttcatggtca gattgtaaag aagcaattac tcttggacag
     2641 tttaagcaga aaagccacca agtcaagtca ctggtcatat gatgaactcg aagatctaat
     2701 aaaagttaat ttaagcgttc attacaataa attcaagtga atctgtttta agctagcctt
     2761 taacgttttt aaattccttc aaacaaatta gttcctcttt gcagttcctc atttctcggt
     2821 gagctggtgc tttaatccct ttactttttc ttcctctagg gagtgatggt tggcatggga
     2881 cgaattaatt cctgcagccc gggggtaccg gtcgccacca tgaacacccc gggaattaac
     2941 ctgatcaagg aggacatgcg cgtgaaggtg cacatggagg gcaacgtgaa cggccacgcc
     3001 ttcgtgatcg agggcgaggg caagggcaag ccctacgagg gcacccagac cgccaacctg
     3061 accgtgaagg agggcgcccc cctgcccttc agctacgaca tcctgaccac cgccgtgcac
     3121 tacggcaacc gggtgttcac caagtacccc gaggacatcc ccgactactt caagcagagc
     3181 ttccccgagg gctacagctg ggagcgcacc atgaccttcg aggacaaggg catctgcacc
     3241 atccgcagcg acatcagcct ggagggcgac tgcttcttcc agaacgtgcg cttcaagggc
     3301 accaacttcc cccccaacgg ccccgtgatg cagaagaaaa ccctgaagtg ggagcccagc
     3361 accgagaagc tgcacgtgcg cgacggcctg ctggtgggca acatcaacat ggccctgctg
     3421 ctggagggcg gcggccacta cctgtgcgac ttcaagacca cctacaaggc caagaaggtg
     3481 gtgcagctgc ccgacgccca cttcgtggac caccgcatcg agatcctggg caacgacagc
     3541 gactacaaca aggtgaagct gtacgagcac gccgtggccc gctacagccc cctgcccagc
     3601 caggtgtggt aatctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     3661 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     3721 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3781 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3841 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3901 atcagctcat tttttaacca ataggccgaa atcggcggat cctgcggccg caagggaaaa
     3961 tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     4021 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     4081 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     4141 aaattactca agtactttac acctctggtt cttgaccccc taccttcagc aagcccagca
     4201 gatccactag atcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     6421 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6481 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     6541 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     6601 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     6661 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6721 agagcttgac ggggaaagcc ggccacaatg acggctacta catggtgcca ttccttcctc
     6781 tttataggaa tggagactac ctcctgtcca acaatgctct tggatacgag tacgcctacc
     6841 tgttggaccc aggtcattgc acaacaccag aaatgccctc tgatctgcaa aagacgtgaa
     6901 tatctgttca gacacccata tccactctgt tccacacagg tcagaggttt gtccaggagt
     6961 tcttgacaga ggtgtaaaaa gtactcaaaa attttactca agtgaaagta caagtactta
     7021 gggaaaattt tactcaatta aaagtaaaag tatctggcta gaatcttact tgagtaaaag
     7081 taaaaaagta ctccattaaa attgtacttg agtattaagg aagtaaaagg ctagcaagat
     7141 ctgctcgaga tacatttgat tagaagagtt tgacttgggt tgattatagc aaatagtgga
     7201 acacaaacag attttgaaat attcttgcaa atttatttat taaatccatt caaatattgt
     7261 cattttttgt actgtgtaat ttatattgat ttgctcatac aatatttaca tttcgcttaa
     7321 acaatacact attatttata tttaaaaata gctatatata aatcaaatgt atagctcttt
     7381 tttacacaat taatcaaagc taaaatattt gtcacacaaa tacagtaagc cacttcacaa
     7441 tgatatcttc aaaatctgtg cttgactgaa tcatttcatt tattgttttg taattcacat
     7501 tataccttac gttctccagg cacagtttga attgttgata aatgaagata atacatctga
     7561 tttaattagt ctcctttgtt tataataact ttaatctaat ttgatagtaa taattccagc
     7621 aatacatcca ttacaataaa aatcgctttt ttattttgca cattagtttg ccggtatttg
     7681 tttaaagttt caccatactt ttaaatcatg tgctatttga aaagggctgt tatgactgga
     7741 attaaatgtt tttttttata tcagtgtttt agattgaa
//
