LOCUS       sequence-182668-        7724 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 7724)
  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 7724)
  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 375-1-Dendra2
FEATURES             Location/Qualifiers
     source          1..7724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1717..1726
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2817..3509
                     /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    3620..3741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3657..3676)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3711..3730
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3865..4052,4053..4064)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4119..4139)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4119..4137)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4158..4174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4158..4174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4171..4193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4182..4198
                     /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(4206..4212,4213..4230,4231..4236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4251..4272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4389..4406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4560..5148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4640..4659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5319..6110,6111..6179))
                     /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     5942..5961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6180..6284)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6447..6468)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6864..6875,6876..7013)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tgctccaggt ccttgtgtcc ttctaaggtc tgtttgtggt ttaagctgtt tgttgacaca
       61 cgcttgtcat ctccattttt ctgtgaagtt attgtcagtt tattgcacac tgggttgcat
      121 taagctgcag ttttttcaca acagttttcc acattgtaac agtcagtctc caaattattc
      181 catcagtcct caaatttagt catttgaggc catgaaaaca cttaaaacta catgaaggta
      241 gtttggcatt aggttttaga cttgttcttg tttggcatat tgcaacctgt gtaaaaatga
      301 tttgattttt attttttttt tttttttttc agaaagaaac ggtccaaaaa gagttatgtg
      361 aacatttatc catgtgttta atgtttttta aattaaaaaa aaacattttt attaaaccac
      421 ttgtgactaa acctttttta gttattctat tttgtgacga gtctgaatgg ccaaagtact
      481 cttgaatcca aaatctttat agtaaaatgt ttaatcttac ttgcagtgtt gggtataatt
      541 tgtaacaaag ttgaattcca tatctgctga gtgaaaaagt aaagtaagag attgctgtaa
      601 atatttaggt tatttattta ttaatacaat atagaaagta acaaaacaca atgtaaaaaa
      661 tcaataaaga aagtaaatat taaaagtaat gataaactaa tgatgtaaac aatattagca
      721 acaaagtttg tacaattcag ggagggtaaa gaatttcacc taatagtcaa actctgtatt
      781 aaataatata aaatctttgt tcttagtctg aagaggagac tgtgagagaa agcttactgg
      841 ttgttaaatt agaaaatttg taacttgcag attaaaatga tttaaataat tttataaagg
      901 ccatgtttga aacaaatgta aaaacataat aaatgttaag aatttcagat attgagattt
      961 tacaatttaa aaaaagcaat aaagtaatag cagagaccta cactcttaaa gtaaattacc
     1021 caactctgct tactagtaac ttttggaata gttactagta tccattgaac aaatactaga
     1081 atctaattaa attgtgtgag tgtccaatga aaagattctt gtatttaaaa cagtaccagt
     1141 atctatatat gtacaaaatt tagtgctagc aaaatgcaaa taaatactac tgcatataat
     1201 ggattgcatt gtaaatcgac tcgccataat acatttattt cctattttta cttttaacaa
     1261 aaaataagtt cactgagtat ttcatgtcat taaatcgcat taaacctctt gcatgcttta
     1321 tgtacactag tgcagttcgc taaatgacca ccggtgtcac taataacacg atcgtttctg
     1381 aattctgcat taaacgccat ttatacgttg ctcttattag taaacaattt ctccatcatg
     1441 agctcgacca gtattgcata tttcatcctc ttaaatccag agttgtttct tttaatcact
     1501 ttctcttcat gataaaagca gtttttggtt ttcagaagtt tacaacaaac ccagtcagtt
     1561 attgcctcag caaacactgg agcaactgca cacttgtgaa aagcttcttg cgaaacacaa
     1621 acctttctct ggacttataa ataaaggcat tcagcagctc caccatacac ctccactggt
     1681 cttgataaag cagatatcag cagtgataca ggtaccgcca ccatggatga ggaaatcgct
     1741 gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg agatgatgcc
     1801 cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca tttaataaac
     1861 catcctaata acatgttaat catattgaac cacattaaac tctcatttgt gcattttaca
     1921 tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag tgttaattca
     1981 cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac ttgatctgat
     2041 tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca cagccatgcc
     2101 atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt ttatgctgtg
     2161 tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta tagtcagtgt
     2221 caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg gcttggagtt
     2281 tcccagtgag cccacagaac ttcgatgaat aaaactgcac ttgctttacg ttgagccaca
     2341 cgcacaatac atgtggattc agttttgttc gttcggctcg cgttaagcaa gtgcagaact
     2401 tcttcagtgc agtttttgga ccacagccaa ctgctgttac tctcgcccag aaatctgcgc
     2461 caacatggac acacccagtg gagcaagcca tctgattttc atggtcagat tgtaaagaag
     2521 caattactct tggacagttt aagcagaaaa gccaccaagt caagtcactg gtcatatgat
     2581 gaactcgaag atctaataaa agttaattta agcgttcatt acaataaatt caagtgaatc
     2641 tgttttaagc tagcctttaa cgtttttaaa ttccttcaaa caaattagtt cctctttgca
     2701 gttcctcatt tctcggtgag ctggtgcttt aatcccttta ctttttcttc ctctagggag
     2761 tgatggttgg catgggacga attaattcct gcagcccggg ggtaccggtc gccaccatga
     2821 acaccccggg aattaacctg atcaaggagg acatgcgcgt gaaggtgcac atggagggca
     2881 acgtgaacgg ccacgccttc gtgatcgagg gcgagggcaa gggcaagccc tacgagggca
     2941 cccagaccgc caacctgacc gtgaaggagg gcgcccccct gcccttcagc tacgacatcc
     3001 tgaccaccgc cgtgcactac ggcaaccggg tgttcaccaa gtaccccgag gacatccccg
     3061 actacttcaa gcagagcttc cccgagggct acagctggga gcgcaccatg accttcgagg
     3121 acaagggcat ctgcaccatc cgcagcgaca tcagcctgga gggcgactgc ttcttccaga
     3181 acgtgcgctt caagggcacc aacttccccc ccaacggccc cgtgatgcag aagaaaaccc
     3241 tgaagtggga gcccagcacc gagaagctgc acgtgcgcga cggcctgctg gtgggcaaca
     3301 tcaacatggc cctgctgctg gagggcggcg gccactacct gtgcgacttc aagaccacct
     3361 acaaggccaa gaaggtggtg cagctgcccg acgcccactt cgtggaccac cgcatcgaga
     3421 tcctgggcaa cgacagcgac tacaacaagg tgaagctgta cgagcacgcc gtggcccgct
     3481 acagccccct gcccagccag gtgtggtaat ctagatcata atcagccata ccacatttgt
     3541 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3601 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3661 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3721 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     3781 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcggatcct
     3841 gcggccgcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta
     3901 aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag
     3961 taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta
     4021 agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac
     4081 cttcagcaag cccagcagat ccactagatc ttttgttccc tttagtgagg gttaattgcg
     4141 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4201 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4261 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4321 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4381 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4441 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4501 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4561 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4621 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4681 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4741 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4801 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4861 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4921 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4981 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5041 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5101 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5161 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5221 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5281 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5341 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5401 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5461 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5521 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5581 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5641 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5701 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5761 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5821 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5881 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5941 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6001 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6061 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6121 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6181 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6241 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6301 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6361 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6421 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6481 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6541 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6601 aagggagccc ccgatttaga gcttgacggg gaaagccggc cacaatgacg gctactacat
     6661 ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca atgctcttgg
     6721 atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga
     6781 tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca
     6841 gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt
     6901 gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa
     6961 tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag
     7021 taaaaggcta gcaagatctg ctcgagatac atttgattag aagagtttga cttgggttga
     7081 ttatagcaaa tagtggaaca caaacagatt ttgaaatatt cttgcaaatt tatttattaa
     7141 atccattcaa atattgtcat tttttgtact gtgtaattta tattgatttg ctcatacaat
     7201 atttacattt cgcttaaaca atacactatt atttatattt aaaaatagct atatataaat
     7261 caaatgtata gctctttttt acacaattaa tcaaagctaa aatatttgtc acacaaatac
     7321 agtaagccac ttcacaatga tatcttcaaa atctgtgctt gactgaatca tttcatttat
     7381 tgttttgtaa ttcacattat accttacgtt ctccaggcac agtttgaatt gttgataaat
     7441 gaagataata catctgattt aattagtctc ctttgtttat aataacttta atctaatttg
     7501 atagtaataa ttccagcaat acatccatta caataaaaat cgctttttta ttttgcacat
     7561 tagtttgccg gtatttgttt aaagtttcac catactttta aatcatgtgc tatttgaaaa
     7621 gggctgttat gactggaatt aaatgttttt ttttatatca gtgttttaga ttgaagtaca
     7681 atatttatag gtaaaattca ccagcatatg cttaaagata gcca
//
