LOCUS       sequence-182666-        7964 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 7964)
  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 7964)
  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 218a-2-Dendra2
FEATURES             Location/Qualifiers
     source          1..7964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1346..1355
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2686..3378
                     /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    3489..3610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3526..3545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3580..3599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3734..3921,3922..3933)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3988..4008)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3988..4006)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4027..4043)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4027..4043)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4040..4062)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4051..4067
                     /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(4075..4081,4082..4099,4100..4105))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4120..4141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4258..4275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4429..5017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4509..4528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5188..5979,5980..6048))
                     /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     5811..5830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6049..6153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6316..6337)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6733..6744,6745..6882)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 atgtgtttaa tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa
       61 ccttttttag ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa
      121 aatctttata gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt
      181 tgaattccat atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt
      241 atttatttat taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa
      301 agtaaatatt aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt
      361 acaattcagg gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa
      421 aatctttgtt cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta
      481 gaaaatttgt aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa
      541 acaaatgtaa aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa
      601 aaaagcaata aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt
      661 actagtaact tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa
      721 ttgtgtgagt gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg
      781 tacaaaattt agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg
      841 taaatcgact cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc
      901 actgagtatt tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt
      961 gcagttcgct aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt
     1021 aaacgccatt tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag
     1081 tattgcatat ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg
     1141 ataaaagcag tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc
     1201 aaacactgga gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg
     1261 gacttataaa taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc
     1321 agatatcagc agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt
     1381 tgacaacggc tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt
     1441 tttcccctcc attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa
     1501 catgttaatc atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac
     1561 cgatttatat tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg
     1621 cttgttttga gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt
     1681 tcgaagttct tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg
     1741 atacagtata agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt
     1801 ttttcagtgg tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt
     1861 atcgtcagcg tgatgggcaa aaaacagctg gcagtacggg ctaccccagc ctatcacaga
     1921 ccaggaaccc aatcacaggg ttcatgcaga tgtgaaaatg agattttagc acatgatgtg
     1981 aactgtatgt gtatgtgttt gtttgactgt ttgtataatc tctgcttcat ctatttctat
     2041 actgcaatgt tcctgccaca atttttccgc ctttctcttg ttttgttttg gcatctttcc
     2101 tctctctgtc atttcaacta gtttacccca gctaaacaaa ttagtatctc attctctcta
     2161 tctttggtct ttccttttct ctgtgtcttg tggaggaaat tcagcggggt tttcctttgt
     2221 gcttgatcta accatgtggt tgcagactca gactaataca tggttctgtc aagcaccatg
     2281 gaaggtcttg cagagccaac tgctgttact ctcgcccaga aatctgcgcc aacatggaca
     2341 cacccagtgg agcaagccat ctgattttca tggtcagatt gtaaagaagc aattactctt
     2401 ggacagttta agcagaaaag ccaccaagtc aagtcactgg tcatatgatg aactcgaaga
     2461 tctaataaaa gttaatttaa gcgttcatta caataaattc aagtgaatct gttttaagct
     2521 agcctttaac gtttttaaat tccttcaaac aaattagttc ctctttgcag ttcctcattt
     2581 ctcggtgagc tggtgcttta atccctttac tttttcttcc tctagggagt gatggttggc
     2641 atgggacgaa ttaattcctg cagcccgggg gtaccggtcg ccaccatgaa caccccggga
     2701 attaacctga tcaaggagga catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc
     2761 cacgccttcg tgatcgaggg cgagggcaag ggcaagccct acgagggcac ccagaccgcc
     2821 aacctgaccg tgaaggaggg cgcccccctg cccttcagct acgacatcct gaccaccgcc
     2881 gtgcactacg gcaaccgggt gttcaccaag taccccgagg acatccccga ctacttcaag
     2941 cagagcttcc ccgagggcta cagctgggag cgcaccatga ccttcgagga caagggcatc
     3001 tgcaccatcc gcagcgacat cagcctggag ggcgactgct tcttccagaa cgtgcgcttc
     3061 aagggcacca acttcccccc caacggcccc gtgatgcaga agaaaaccct gaagtgggag
     3121 cccagcaccg agaagctgca cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc
     3181 ctgctgctgg agggcggcgg ccactacctg tgcgacttca agaccaccta caaggccaag
     3241 aaggtggtgc agctgcccga cgcccacttc gtggaccacc gcatcgagat cctgggcaac
     3301 gacagcgact acaacaaggt gaagctgtac gagcacgccg tggcccgcta cagccccctg
     3361 cccagccagg tgtggtaatc tagatcataa tcagccatac cacatttgta gaggttttac
     3421 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3481 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3541 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3601 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3661 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcggatcctg cggccgcaag
     3721 ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     3781 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     3841 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     3901 caagtaaaat tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc
     3961 ccagcagatc cactagatct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4021 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4081 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4141 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4201 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4261 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4321 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4381 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4441 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4501 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4561 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4621 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4681 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4741 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4801 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4861 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4921 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4981 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5041 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5101 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5161 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5221 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5281 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5341 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5401 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5461 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5521 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5581 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5641 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5701 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5761 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5821 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5881 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5941 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6001 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6061 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6121 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     6181 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6241 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6301 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6361 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6421 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6481 cgatttagag cttgacgggg aaagccggcc acaatgacgg ctactacatg gtgccattcc
     6541 ttcctcttta taggaatgga gactacctcc tgtccaacaa tgctcttgga tacgagtacg
     6601 cctacctgtt ggacccaggt cattgcacaa caccagaaat gccctctgat ctgcaaaaga
     6661 cgtgaatatc tgttcagaca cccatatcca ctctgttcca cacaggtcag aggtttgtcc
     6721 aggagttctt gacagaggtg taaaaagtac tcaaaaattt tactcaagtg aaagtacaag
     6781 tacttaggga aaattttact caattaaaag taaaagtatc tggctagaat cttacttgag
     6841 taaaagtaaa aaagtactcc attaaaattg tacttgagta ttaaggaagt aaaaggctag
     6901 caagatctgc tcgagataca tttgattaga agagtttgac ttgggttgat tatagcaaat
     6961 agtggaacac aaacagattt tgaaatattc ttgcaaattt atttattaaa tccattcaaa
     7021 tattgtcatt ttttgtactg tgtaatttat attgatttgc tcatacaata tttacatttc
     7081 gcttaaacaa tacactatta tttatattta aaaatagcta tatataaatc aaatgtatag
     7141 ctctttttta cacaattaat caaagctaaa atatttgtca cacaaataca gtaagccact
     7201 tcacaatgat atcttcaaaa tctgtgcttg actgaatcat ttcatttatt gttttgtaat
     7261 tcacattata ccttacgttc tccaggcaca gtttgaattg ttgataaatg aagataatac
     7321 atctgattta attagtctcc tttgtttata ataactttaa tctaatttga tagtaataat
     7381 tccagcaata catccattac aataaaaatc gcttttttat tttgcacatt agtttgccgg
     7441 tatttgttta aagtttcacc atacttttaa atcatgtgct atttgaaaag ggctgttatg
     7501 actggaatta aatgtttttt tttatatcag tgttttagat tgaagtacaa tatttatagg
     7561 taaaattcac cagcatatgc ttaaagatag ccatgctcca ggtccttgtg tccttctaag
     7621 gtctgtttgt ggtttaagct gtttgttgac acacgcttgt catctccatt tttctgtgaa
     7681 gttattgtca gtttattgca cactgggttg cattaagctg cagttttttc acaacagttt
     7741 tccacattgt aacagtcagt ctccaaatta ttccatcagt cctcaaattt agtcatttga
     7801 ggccatgaaa acacttaaaa ctacatgaag gtagtttggc attaggtttt agacttgttc
     7861 ttgtttggca tattgcaacc tgtgtaaaaa tgatttgatt tttatttttt tttttttttt
     7921 ttcagaaaga aacggtccaa aaagagttat gtgaacattt atcc
//
