LOCUS       sequence-182670-        8124 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 8124)
  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 8124)
  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 457a-Dendra2
FEATURES             Location/Qualifiers
     source          1..8124
                     /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             3266..3958
                     /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    4069..4190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4106..4125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4160..4179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4314..4501,4502..4513)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4568..4588)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4568..4586)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4607..4623)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4607..4623)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4620..4642)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4631..4647
                     /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(4655..4661,4662..4679,4680..4685))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4700..4721
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4838..4855)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5009..5597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5089..5108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5768..6559,6560..6628))
                     /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     6391..6410
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6629..6733)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6896..6917)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7313..7324,7325..7462)
                     /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 ctgctgaatc accaggtttg
     2341 gctcgcatgg cctcttcaga cagttgggaa ttcacatcct cttttttttt tagtctatat
     2401 tttaagatgt cagctacttt ctgcgacagt gtaaccgttc aagctgatga attaatctgc
     2461 atatatttgt gacgttccca tcatcctctt tcctcccttg gaccacttga tgcctcagtg
     2521 tgcctgacag aagcagcaca tcaatattgg cagctgccct ctctctgggt tgccagtatg
     2581 gtttgtgctg ctcccgtcag acagacacac tcagcgatcc tccagtattt ggagcaatat
     2641 gaaaaccacc tgtccggaaa ttaattgtat gtatttgact ttgacaatgt cactttcaac
     2701 tgaaatgctt tctctttgtt cggacttgca tgaaaaacct aacattaaaa aatattacaa
     2761 acaaaacctt atttgagtga aattattttt attttcaata cacgaaatga aagatatcag
     2821 tggtttcagt ttgtcgctga taaactggtg aaaaccaatg tgtgctgtcg ctaagccaac
     2881 tgctgttact ctcgcccaga aatctgcgcc aacatggaca cacccagtgg agcaagccat
     2941 ctgattttca tggtcagatt gtaaagaagc aattactctt ggacagttta agcagaaaag
     3001 ccaccaagtc aagtcactgg tcatatgatg aactcgaaga tctaataaaa gttaatttaa
     3061 gcgttcatta caataaattc aagtgaatct gttttaagct agcctttaac gtttttaaat
     3121 tccttcaaac aaattagttc ctctttgcag ttcctcattt ctcggtgagc tggtgcttta
     3181 atccctttac tttttcttcc tctagggagt gatggttggc atgggacgaa ttaattcctg
     3241 cagcccgggg gtaccggtcg ccaccatgaa caccccggga attaacctga tcaaggagga
     3301 catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc cacgccttcg tgatcgaggg
     3361 cgagggcaag ggcaagccct acgagggcac ccagaccgcc aacctgaccg tgaaggaggg
     3421 cgcccccctg cccttcagct acgacatcct gaccaccgcc gtgcactacg gcaaccgggt
     3481 gttcaccaag taccccgagg acatccccga ctacttcaag cagagcttcc ccgagggcta
     3541 cagctgggag cgcaccatga ccttcgagga caagggcatc tgcaccatcc gcagcgacat
     3601 cagcctggag ggcgactgct tcttccagaa cgtgcgcttc aagggcacca acttcccccc
     3661 caacggcccc gtgatgcaga agaaaaccct gaagtgggag cccagcaccg agaagctgca
     3721 cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc ctgctgctgg agggcggcgg
     3781 ccactacctg tgcgacttca agaccaccta caaggccaag aaggtggtgc agctgcccga
     3841 cgcccacttc gtggaccacc gcatcgagat cctgggcaac gacagcgact acaacaaggt
     3901 gaagctgtac gagcacgccg tggcccgcta cagccccctg cccagccagg tgtggtaatc
     3961 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     4021 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     4081 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4141 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     4201 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4261 taaccaatag gccgaaatcg gcggatcctg cggccgcaag ggaaaataga atgaagtgat
     4321 ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt
     4381 ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt
     4441 aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta
     4501 ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc cactagatct
     4561 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     4621 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4681 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4741 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4801 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4861 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4921 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4981 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5041 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5101 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5161 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5221 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5281 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5341 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5401 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5461 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5521 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5581 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5641 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5701 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5761 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5821 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5881 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5941 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6001 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6061 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6121 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6181 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6241 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6301 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6361 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6421 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6481 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6541 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6601 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6661 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6721 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6781 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6841 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6901 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6961 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     7021 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     7081 aaagccggcc acaatgacgg ctactacatg gtgccattcc ttcctcttta taggaatgga
     7141 gactacctcc tgtccaacaa tgctcttgga tacgagtacg cctacctgtt ggacccaggt
     7201 cattgcacaa caccagaaat gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca
     7261 cccatatcca ctctgttcca cacaggtcag aggtttgtcc aggagttctt gacagaggtg
     7321 taaaaagtac tcaaaaattt tactcaagtg aaagtacaag tacttaggga aaattttact
     7381 caattaaaag taaaagtatc tggctagaat cttacttgag taaaagtaaa aaagtactcc
     7441 attaaaattg tacttgagta ttaaggaagt aaaaggctag caagatctgc tcgagataca
     7501 tttgattaga agagtttgac ttgggttgat tatagcaaat agtggaacac aaacagattt
     7561 tgaaatattc ttgcaaattt atttattaaa tccattcaaa tattgtcatt ttttgtactg
     7621 tgtaatttat attgatttgc tcatacaata tttacatttc gcttaaacaa tacactatta
     7681 tttatattta aaaatagcta tatataaatc aaatgtatag ctctttttta cacaattaat
     7741 caaagctaaa atatttgtca cacaaataca gtaagccact tcacaatgat atcttcaaaa
     7801 tctgtgcttg actgaatcat ttcatttatt gttttgtaat tcacattata ccttacgttc
     7861 tccaggcaca gtttgaattg ttgataaatg aagataatac atctgattta attagtctcc
     7921 tttgtttata ataactttaa tctaatttga tagtaataat tccagcaata catccattac
     7981 aataaaaatc gcttttttat tttgcacatt agtttgccgg tatttgttta aagtttcacc
     8041 atacttttaa atcatgtgct atttgaaaag ggctgttatg actggaatta aatgtttttt
     8101 tttatatcag tgttttagat tgaa
//
