LOCUS       sequence-182672-        7794 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 7794)
  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 7794)
  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 724-Dendra2
FEATURES             Location/Qualifiers
     source          1..7794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(142..163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(559..570,571..708)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      3136..3145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4306..4998
                     /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    5109..5230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5146..5165)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5200..5219
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(5354..5541,5542..5553)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5608..5628)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5608..5626)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5647..5663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5647..5663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5660..5682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5671..5687
                     /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(5695..5701,5702..5719,5720..5725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5878..5895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6049..6637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6129..6148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6808..7599,7600..7668))
                     /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     7431..7450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7669..7773)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
       61 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      121 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      181 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      241 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      301 agcccccgat ttagagcttg acggggaaag ccggccacaa tgacggctac tacatggtgc
      361 cattccttcc tctttatagg aatggagact acctcctgtc caacaatgct cttggatacg
      421 agtacgccta cctgttggac ccaggtcatt gcacaacacc agaaatgccc tctgatctgc
      481 aaaagacgtg aatatctgtt cagacaccca tatccactct gttccacaca ggtcagaggt
      541 ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      601 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      661 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
      721 ggctagcaag atctgctcga gatacatttg attagaagag tttgacttgg gttgattata
      781 gcaaatagtg gaacacaaac agattttgaa atattcttgc aaatttattt attaaatcca
      841 ttcaaatatt gtcatttttt gtactgtgta atttatattg atttgctcat acaatattta
      901 catttcgctt aaacaataca ctattattta tatttaaaaa tagctatata taaatcaaat
      961 gtatagctct tttttacaca attaatcaaa gctaaaatat ttgtcacaca aatacagtaa
     1021 gccacttcac aatgatatct tcaaaatctg tgcttgactg aatcatttca tttattgttt
     1081 tgtaattcac attatacctt acgttctcca ggcacagttt gaattgttga taaatgaaga
     1141 taatacatct gatttaatta gtctcctttg tttataataa ctttaatcta atttgatagt
     1201 aataattcca gcaatacatc cattacaata aaaatcgctt ttttattttg cacattagtt
     1261 tgccggtatt tgtttaaagt ttcaccatac ttttaaatca tgtgctattt gaaaagggct
     1321 gttatgactg gaattaaatg ttttttttta tatcagtgtt ttagattgaa gtacaatatt
     1381 tataggtaaa attcaccagc atatgcttaa agatagccat gctccaggtc cttgtgtcct
     1441 tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc tccatttttc
     1501 tgtgaagtta ttgtcagttt attgcacact gggttgcatt aagctgcagt tttttcacaa
     1561 cagttttcca cattgtaaca gtcagtctcc aaattattcc atcagtcctc aaatttagtc
     1621 atttgaggcc atgaaaacac ttaaaactac atgaaggtag tttggcatta ggttttagac
     1681 ttgttcttgt ttggcatatt gcaacctgtg taaaaatgat ttgattttta tttttttttt
     1741 ttttttttca gaaagaaacg gtccaaaaag agttatgtga acatttatcc atgtgtttaa
     1801 tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa ccttttttag
     1861 ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa aatctttata
     1921 gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt tgaattccat
     1981 atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt atttatttat
     2041 taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa agtaaatatt
     2101 aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt acaattcagg
     2161 gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa aatctttgtt
     2221 cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta gaaaatttgt
     2281 aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa acaaatgtaa
     2341 aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa aaaagcaata
     2401 aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt actagtaact
     2461 tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa ttgtgtgagt
     2521 gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg tacaaaattt
     2581 agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg taaatcgact
     2641 cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc actgagtatt
     2701 tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt gcagttcgct
     2761 aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt aaacgccatt
     2821 tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag tattgcatat
     2881 ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg ataaaagcag
     2941 tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc aaacactgga
     3001 gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg gacttataaa
     3061 taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc agatatcagc
     3121 agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt tgacaacggc
     3181 tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt tttcccctcc
     3241 attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa catgttaatc
     3301 atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac cgatttatat
     3361 tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg cttgttttga
     3421 gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt tcgaagttct
     3481 tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg atacagtata
     3541 agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt ttttcagtgg
     3601 tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt atcgtcagcg
     3661 tgatgggcaa aaaacagctg gcagtacggg ctagccaatc aacaggctcg atttcccagc
     3721 agactggatt taaagggaat ttgcgactgt tagtcagatt tgtagaacag ccacaccttc
     3781 cttttaagat cttgcctgct gctctttccc caacggcctg catctgccaa gaagactctt
     3841 tcacagcgtt tacatccctg cgtgactttt cagacctgga tgcaaaaggt aaaaacgaag
     3901 acctgtgtgt ggtagccaac tgctgttact ctcgcccaga aatctgcgcc aacatggaca
     3961 cacccagtgg agcaagccat ctgattttca tggtcagatt gtaaagaagc aattactctt
     4021 ggacagttta agcagaaaag ccaccaagtc aagtcactgg tcatatgatg aactcgaaga
     4081 tctaataaaa gttaatttaa gcgttcatta caataaattc aagtgaatct gttttaagct
     4141 agcctttaac gtttttaaat tccttcaaac aaattagttc ctctttgcag ttcctcattt
     4201 ctcggtgagc tggtgcttta atccctttac tttttcttcc tctagggagt gatggttggc
     4261 atgggacgaa ttaattcctg cagcccgggg gtaccggtcg ccaccatgaa caccccggga
     4321 attaacctga tcaaggagga catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc
     4381 cacgccttcg tgatcgaggg cgagggcaag ggcaagccct acgagggcac ccagaccgcc
     4441 aacctgaccg tgaaggaggg cgcccccctg cccttcagct acgacatcct gaccaccgcc
     4501 gtgcactacg gcaaccgggt gttcaccaag taccccgagg acatccccga ctacttcaag
     4561 cagagcttcc ccgagggcta cagctgggag cgcaccatga ccttcgagga caagggcatc
     4621 tgcaccatcc gcagcgacat cagcctggag ggcgactgct tcttccagaa cgtgcgcttc
     4681 aagggcacca acttcccccc caacggcccc gtgatgcaga agaaaaccct gaagtgggag
     4741 cccagcaccg agaagctgca cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc
     4801 ctgctgctgg agggcggcgg ccactacctg tgcgacttca agaccaccta caaggccaag
     4861 aaggtggtgc agctgcccga cgcccacttc gtggaccacc gcatcgagat cctgggcaac
     4921 gacagcgact acaacaaggt gaagctgtac gagcacgccg tggcccgcta cagccccctg
     4981 cccagccagg tgtggtaatc tagatcataa tcagccatac cacatttgta gaggttttac
     5041 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     5101 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5161 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5221 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     5281 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcggatcctg cggccgcaag
     5341 ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     5401 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     5461 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     5521 caagtaaaat tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc
     5581 ccagcagatc cactagatct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     5641 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5701 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5881 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5941 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6001 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6061 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6121 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6181 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6241 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6301 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6361 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6421 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6481 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6541 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6601 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6661 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6721 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6781 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6841 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6901 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6961 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7021 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7081 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7141 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7201 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7261 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7321 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7381 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7441 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7501 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7561 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7621 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7681 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7741 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgcc
//
