LOCUS       sequence-182578-        7795 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 7795)
  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 7795)
  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 24-1-Dendra2
FEATURES             Location/Qualifiers
     source          1..7795
                     /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             2937..3629
                     /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    3740..3861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3777..3796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3831..3850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3985..4172,4173..4184)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4239..4259)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4239..4257)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4278..4294)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4278..4294)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4291..4313)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4302..4318
                     /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(4326..4332,4333..4350,4351..4356))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4371..4392
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4509..4526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4680..5268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4760..4779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5439..6230,6231..6299))
                     /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     6062..6081
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6300..6404)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6567..6588)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6984..6995,6996..7133)
                     /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 ctaagcccat gaccaaggta
     2341 agagttaagg tttctaactt actcctttct ttccacacac ctggtcatct gatgtcagct
     2401 gataatctgt ttggagttgt gtgtctcatc aggacctgag ctccggtgcc ttctgagctg
     2461 atatcagttg tagtaaatca ctggctcagt tcagcaggaa caggagtgtg gcccctccac
     2521 aaagaagcac acattctgga gcgtagccaa ctgctgttac tctcgcccag aaatctgcgc
     2581 caacatggac acacccagtg gagcaagcca tctgattttc atggtcagat tgtaaagaag
     2641 caattactct tggacagttt aagcagaaaa gccaccaagt caagtcactg gtcatatgat
     2701 gaactcgaag atctaataaa agttaattta agcgttcatt acaataaatt caagtgaatc
     2761 tgttttaagc tagcctttaa cgtttttaaa ttccttcaaa caaattagtt cctctttgca
     2821 gttcctcatt tctcggtgag ctggtgcttt aatcccttta ctttttcttc ctctagggag
     2881 tgatggttgg catgggacga attaattcct gcagcccggg ggtaccggtc gccaccatga
     2941 acaccccggg aattaacctg atcaaggagg acatgcgcgt gaaggtgcac atggagggca
     3001 acgtgaacgg ccacgccttc gtgatcgagg gcgagggcaa gggcaagccc tacgagggca
     3061 cccagaccgc caacctgacc gtgaaggagg gcgcccccct gcccttcagc tacgacatcc
     3121 tgaccaccgc cgtgcactac ggcaaccggg tgttcaccaa gtaccccgag gacatccccg
     3181 actacttcaa gcagagcttc cccgagggct acagctggga gcgcaccatg accttcgagg
     3241 acaagggcat ctgcaccatc cgcagcgaca tcagcctgga gggcgactgc ttcttccaga
     3301 acgtgcgctt caagggcacc aacttccccc ccaacggccc cgtgatgcag aagaaaaccc
     3361 tgaagtggga gcccagcacc gagaagctgc acgtgcgcga cggcctgctg gtgggcaaca
     3421 tcaacatggc cctgctgctg gagggcggcg gccactacct gtgcgacttc aagaccacct
     3481 acaaggccaa gaaggtggtg cagctgcccg acgcccactt cgtggaccac cgcatcgaga
     3541 tcctgggcaa cgacagcgac tacaacaagg tgaagctgta cgagcacgcc gtggcccgct
     3601 acagccccct gcccagccag gtgtggtaat ctagatcata atcagccata ccacatttgt
     3661 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3721 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3781 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3841 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     3901 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcggatcct
     3961 gcggccgcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta
     4021 aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag
     4081 taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta
     4141 agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac
     4201 cttcagcaag cccagcagat ccactagatc ttttgttccc tttagtgagg gttaattgcg
     4261 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4321 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4381 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4441 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4501 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4561 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4621 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4681 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4741 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4801 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4861 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4921 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4981 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5041 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5101 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5161 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5221 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5281 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5341 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5401 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5461 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5521 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5581 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5641 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5701 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5761 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5821 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5881 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5941 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6001 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6061 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6121 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6181 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6241 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6301 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6361 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6421 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6481 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6541 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6601 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6661 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6721 aagggagccc ccgatttaga gcttgacggg gaaagccggc cacaatgacg gctactacat
     6781 ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca atgctcttgg
     6841 atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga
     6901 tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca
     6961 gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt
     7021 gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa
     7081 tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag
     7141 taaaaggcta gcaagatctg ctcgagatac atttgattag aagagtttga cttgggttga
     7201 ttatagcaaa tagtggaaca caaacagatt ttgaaatatt cttgcaaatt tatttattaa
     7261 atccattcaa atattgtcat tttttgtact gtgtaattta tattgatttg ctcatacaat
     7321 atttacattt cgcttaaaca atacactatt atttatattt aaaaatagct atatataaat
     7381 caaatgtata gctctttttt acacaattaa tcaaagctaa aatatttgtc acacaaatac
     7441 agtaagccac ttcacaatga tatcttcaaa atctgtgctt gactgaatca tttcatttat
     7501 tgttttgtaa ttcacattat accttacgtt ctccaggcac agtttgaatt gttgataaat
     7561 gaagataata catctgattt aattagtctc ctttgtttat aataacttta atctaatttg
     7621 atagtaataa ttccagcaat acatccatta caataaaaat cgctttttta ttttgcacat
     7681 tagtttgccg gtatttgttt aaagtttcac catactttta aatcatgtgc tatttgaaaa
     7741 gggctgttat gactggaatt aaatgttttt ttttatatca gtgttttaga ttgaa
//
