LOCUS       sequence-182761-        7965 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 7965)
  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 7965)
  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 727-Dendra2
FEATURES             Location/Qualifiers
     source          1..7965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1333..1342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2674..3366
                     /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    3477..3598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3514..3533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3568..3587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3722..3909,3910..3921)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3976..3996)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3976..3994)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4015..4031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4015..4031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4028..4050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4039..4055
                     /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(4063..4069,4070..4087,4088..4093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4108..4129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4246..4263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4417..5005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4497..4516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5176..5967,5968..6036))
                     /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     5799..5818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6037..6141)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6304..6325)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6721..6732,6733..6870)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tttttaaatt aaaaaaaaac atttttatta aaccacttgt gactaaacct tttttagtta
       61 ttctattttg tgacgagtct gaatggccaa agtactcttg aatccaaaat ctttatagta
      121 aaatgtttaa tcttacttgc agtgttgggt ataatttgta acaaagttga attccatatc
      181 tgctgagtga aaaagtaaag taagagattg ctgtaaatat ttaggttatt tatttattaa
      241 tacaatatag aaagtaacaa aacacaatgt aaaaaatcaa taaagaaagt aaatattaaa
      301 agtaatgata aactaatgat gtaaacaata ttagcaacaa agtttgtaca attcagggag
      361 ggtaaagaat ttcacctaat agtcaaactc tgtattaaat aatataaaat ctttgttctt
      421 agtctgaaga ggagactgtg agagaaagct tactggttgt taaattagaa aatttgtaac
      481 ttgcagatta aaatgattta aataatttta taaaggccat gtttgaaaca aatgtaaaaa
      541 cataataaat gttaagaatt tcagatattg agattttaca atttaaaaaa agcaataaag
      601 taatagcaga gacctacact cttaaagtaa attacccaac tctgcttact agtaactttt
      661 ggaatagtta ctagtatcca ttgaacaaat actagaatct aattaaattg tgtgagtgtc
      721 caatgaaaag attcttgtat ttaaaacagt accagtatct atatatgtac aaaatttagt
      781 gctagcaaaa tgcaaataaa tactactgca tataatggat tgcattgtaa atcgactcgc
      841 cataatacat ttatttccta tttttacttt taacaaaaaa taagttcact gagtatttca
      901 tgtcattaaa tcgcattaaa cctcttgcat gctttatgta cactagtgca gttcgctaaa
      961 tgaccaccgg tgtcactaat aacacgatcg tttctgaatt ctgcattaaa cgccatttat
     1021 acgttgctct tattagtaaa caatttctcc atcatgagct cgaccagtat tgcatatttc
     1081 atcctcttaa atccagagtt gtttctttta atcactttct cttcatgata aaagcagttt
     1141 ttggttttca gaagtttaca acaaacccag tcagttattg cctcagcaaa cactggagca
     1201 actgcacact tgtgaaaagc ttcttgcgaa acacaaacct ttctctggac ttataaataa
     1261 aggcattcag cagctccacc atacacctcc actggtcttg ataaagcaga tatcagcagt
     1321 gatacaggta ccgccaccat ggatgaggaa atcgctgccc tggtcgttga caacggctcc
     1381 ggtatgtgca aagccggttt tgctggagat gatgcccctc gtgctgtttt cccctccatt
     1441 gttggacgac ccagacatca ggtacattta ataaaccatc ctaataacat gttaatcata
     1501 ttgaaccaca ttaaactctc atttgtgcat tttacatctt ttaaaaccga tttatattcg
     1561 tttttagata atgaagttta ttaaagtgtt aattcacagc tttattgctt gttttgagaa
     1621 tctgcttgca taagtgattt ttaaacttga tctgattttc gtttgtttcg aagttcttca
     1681 gctttgaaag tcagatcttc aattcacagc catgccatag ttttaggata cagtataagt
     1741 agacgaattg ggatgcatcg tagattttat gctgtgtttg ttatggtttt tcagtggtag
     1801 agattttcag agtattatta gggttatagt cagtgtcagg tttatttatc gtcagcgtga
     1861 tgggcaaaaa acagctggca gtacgggctt cctgaacttg tacacatgcc attgtgtttg
     1921 taatttcttt gcgctttcct tctgtatgga tttgagcttc tgctccaaac agttgcttga
     1981 gataagagtg aagatgaaga cttggcgaaa cgggtttttc cagtcacctg tatgtcattt
     2041 tcagtcttca attcctccca gcccgtaccc atcgaaactg tgagttgagg cgagttgaag
     2101 acttaaagtg ctgtacagat tcaagtatct gctcggcatg tgtcgatatg accaacggaa
     2161 attgaaagac agcgaaaatg gaaaaatata tactgtatat atacagtcag ttttgcagtg
     2221 gcaaagcccc tgaagtggtg agtaaataac tgtactgtta tacaggacga gttcaggagc
     2281 tagccaactg ctgttactct cgcccagaaa tctgcgccaa catggacaca cccagtggag
     2341 caagccatct gattttcatg gtcagattgt aaagaagcaa ttactcttgg acagtttaag
     2401 cagaaaagcc accaagtcaa gtcactggtc atatgatgaa ctcgaagatc taataaaagt
     2461 taatttaagc gttcattaca ataaattcaa gtgaatctgt tttaagctag cctttaacgt
     2521 ttttaaattc cttcaaacaa attagttcct ctttgcagtt cctcatttct cggtgagctg
     2581 gtgctttaat ccctttactt tttcttcctc tagggagtga tggttggcat gggacgaatt
     2641 aattcctgca gcccgggggt accggtcgcc accatgaaca ccccgggaat taacctgatc
     2701 aaggaggaca tgcgcgtgaa ggtgcacatg gagggcaacg tgaacggcca cgccttcgtg
     2761 atcgagggcg agggcaaggg caagccctac gagggcaccc agaccgccaa cctgaccgtg
     2821 aaggagggcg cccccctgcc cttcagctac gacatcctga ccaccgccgt gcactacggc
     2881 aaccgggtgt tcaccaagta ccccgaggac atccccgact acttcaagca gagcttcccc
     2941 gagggctaca gctgggagcg caccatgacc ttcgaggaca agggcatctg caccatccgc
     3001 agcgacatca gcctggaggg cgactgcttc ttccagaacg tgcgcttcaa gggcaccaac
     3061 ttccccccca acggccccgt gatgcagaag aaaaccctga agtgggagcc cagcaccgag
     3121 aagctgcacg tgcgcgacgg cctgctggtg ggcaacatca acatggccct gctgctggag
     3181 ggcggcggcc actacctgtg cgacttcaag accacctaca aggccaagaa ggtggtgcag
     3241 ctgcccgacg cccacttcgt ggaccaccgc atcgagatcc tgggcaacga cagcgactac
     3301 aacaaggtga agctgtacga gcacgccgtg gcccgctaca gccccctgcc cagccaggtg
     3361 tggtaatcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     3421 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     3481 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3541 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     3601 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3661 tcatttttta accaataggc cgaaatcggc ggatcctgcg gccgcaaggg aaaatagaat
     3721 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa
     3781 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt
     3841 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta
     3901 ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc agcagatcca
     3961 ctagatcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     4021 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4081 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4141 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4201 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4261 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4321 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4381 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4441 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4501 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4561 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4621 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4681 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4741 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4801 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4861 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4921 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4981 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5041 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5101 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5161 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5221 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5281 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5341 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5401 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5461 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5521 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5581 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5641 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5701 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5761 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5821 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5881 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5941 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6001 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6061 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6121 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     6181 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6241 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6301 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6361 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6421 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6481 tgacggggaa agccggccac aatgacggct actacatggt gccattcctt cctctttata
     6541 ggaatggaga ctacctcctg tccaacaatg ctcttggata cgagtacgcc tacctgttgg
     6601 acccaggtca ttgcacaaca ccagaaatgc cctctgatct gcaaaagacg tgaatatctg
     6661 ttcagacacc catatccact ctgttccaca caggtcagag gtttgtccag gagttcttga
     6721 cagaggtgta aaaagtactc aaaaatttta ctcaagtgaa agtacaagta cttagggaaa
     6781 attttactca attaaaagta aaagtatctg gctagaatct tacttgagta aaagtaaaaa
     6841 agtactccat taaaattgta cttgagtatt aaggaagtaa aaggctagca agatctgctc
     6901 gagatacatt tgattagaag agtttgactt gggttgatta tagcaaatag tggaacacaa
     6961 acagattttg aaatattctt gcaaatttat ttattaaatc cattcaaata ttgtcatttt
     7021 ttgtactgtg taatttatat tgatttgctc atacaatatt tacatttcgc ttaaacaata
     7081 cactattatt tatatttaaa aatagctata tataaatcaa atgtatagct cttttttaca
     7141 caattaatca aagctaaaat atttgtcaca caaatacagt aagccacttc acaatgatat
     7201 cttcaaaatc tgtgcttgac tgaatcattt catttattgt tttgtaattc acattatacc
     7261 ttacgttctc caggcacagt ttgaattgtt gataaatgaa gataatacat ctgatttaat
     7321 tagtctcctt tgtttataat aactttaatc taatttgata gtaataattc cagcaataca
     7381 tccattacaa taaaaatcgc ttttttattt tgcacattag tttgccggta tttgtttaaa
     7441 gtttcaccat acttttaaat catgtgctat ttgaaaaggg ctgttatgac tggaattaaa
     7501 tgtttttttt tatatcagtg ttttagattg aagtacaata tttataggta aaattcacca
     7561 gcatatgctt aaagatagcc atgctccagg tccttgtgtc cttctaaggt ctgtttgtgg
     7621 tttaagctgt ttgttgacac acgcttgtca tctccatttt tctgtgaagt tattgtcagt
     7681 ttattgcaca ctgggttgca ttaagctgca gttttttcac aacagttttc cacattgtaa
     7741 cagtcagtct ccaaattatt ccatcagtcc tcaaatttag tcatttgagg ccatgaaaac
     7801 acttaaaact acatgaaggt agtttggcat taggttttag acttgttctt gtttggcata
     7861 ttgcaacctg tgtaaaaatg atttgatttt tatttttttt tttttttttt cagaaagaaa
     7921 cggtccaaaa agagttatgt gaacatttat ccatgtgttt aatgt
//
