LOCUS       sequence-182667-        7939 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 7939)
  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 7939)
  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 301a-Dendra2
FEATURES             Location/Qualifiers
     source          1..7939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1722..1731
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3036..3728
                     /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    3839..3960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3876..3895)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3930..3949
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(4084..4271,4272..4283)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4338..4358)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4338..4356)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4377..4393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4377..4393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4390..4412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4401..4417
                     /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(4425..4431,4432..4449,4450..4455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4608..4625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4779..5367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4859..4878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5538..6329,6330..6398))
                     /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     6161..6180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6399..6503)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6666..6687)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(7083..7094,7095..7232)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gccatgctcc aggtccttgt gtccttctaa ggtctgtttg tggtttaagc tgtttgttga
       61 cacacgcttg tcatctccat ttttctgtga agttattgtc agtttattgc acactgggtt
      121 gcattaagct gcagtttttt cacaacagtt ttccacattg taacagtcag tctccaaatt
      181 attccatcag tcctcaaatt tagtcatttg aggccatgaa aacacttaaa actacatgaa
      241 ggtagtttgg cattaggttt tagacttgtt cttgtttggc atattgcaac ctgtgtaaaa
      301 atgatttgat ttttattttt tttttttttt ttttcagaaa gaaacggtcc aaaaagagtt
      361 atgtgaacat ttatccatgt gtttaatgtt ttttaaatta aaaaaaaaca tttttattaa
      421 accacttgtg actaaacctt ttttagttat tctattttgt gacgagtctg aatggccaaa
      481 gtactcttga atccaaaatc tttatagtaa aatgtttaat cttacttgca gtgttgggta
      541 taatttgtaa caaagttgaa ttccatatct gctgagtgaa aaagtaaagt aagagattgc
      601 tgtaaatatt taggttattt atttattaat acaatataga aagtaacaaa acacaatgta
      661 aaaaatcaat aaagaaagta aatattaaaa gtaatgataa actaatgatg taaacaatat
      721 tagcaacaaa gtttgtacaa ttcagggagg gtaaagaatt tcacctaata gtcaaactct
      781 gtattaaata atataaaatc tttgttctta gtctgaagag gagactgtga gagaaagctt
      841 actggttgtt aaattagaaa atttgtaact tgcagattaa aatgatttaa ataattttat
      901 aaaggccatg tttgaaacaa atgtaaaaac ataataaatg ttaagaattt cagatattga
      961 gattttacaa tttaaaaaaa gcaataaagt aatagcagag acctacactc ttaaagtaaa
     1021 ttacccaact ctgcttacta gtaacttttg gaatagttac tagtatccat tgaacaaata
     1081 ctagaatcta attaaattgt gtgagtgtcc aatgaaaaga ttcttgtatt taaaacagta
     1141 ccagtatcta tatatgtaca aaatttagtg ctagcaaaat gcaaataaat actactgcat
     1201 ataatggatt gcattgtaaa tcgactcgcc ataatacatt tatttcctat ttttactttt
     1261 aacaaaaaat aagttcactg agtatttcat gtcattaaat cgcattaaac ctcttgcatg
     1321 ctttatgtac actagtgcag ttcgctaaat gaccaccggt gtcactaata acacgatcgt
     1381 ttctgaattc tgcattaaac gccatttata cgttgctctt attagtaaac aatttctcca
     1441 tcatgagctc gaccagtatt gcatatttca tcctcttaaa tccagagttg tttcttttaa
     1501 tcactttctc ttcatgataa aagcagtttt tggttttcag aagtttacaa caaacccagt
     1561 cagttattgc ctcagcaaac actggagcaa ctgcacactt gtgaaaagct tcttgcgaaa
     1621 cacaaacctt tctctggact tataaataaa ggcattcagc agctccacca tacacctcca
     1681 ctggtcttga taaagcagat atcagcagtg atacaggtac cgccaccatg gatgaggaaa
     1741 tcgctgccct ggtcgttgac aacggctccg gtatgtgcaa agccggtttt gctggagatg
     1801 atgcccctcg tgctgttttc ccctccattg ttggacgacc cagacatcag gtacatttaa
     1861 taaaccatcc taataacatg ttaatcatat tgaaccacat taaactctca tttgtgcatt
     1921 ttacatcttt taaaaccgat ttatattcgt ttttagataa tgaagtttat taaagtgtta
     1981 attcacagct ttattgcttg ttttgagaat ctgcttgcat aagtgatttt taaacttgat
     2041 ctgattttcg tttgtttcga agttcttcag ctttgaaagt cagatcttca attcacagcc
     2101 atgccatagt tttaggatac agtataagta gacgaattgg gatgcatcgt agattttatg
     2161 ctgtgtttgt tatggttttt cagtggtaga gattttcaga gtattattag ggttatagtc
     2221 agtgtcaggt ttatttatcg tcagcgtgat gggcaaaaaa cagctggcag tacgggctag
     2281 tggacagcag aggcaatact gatagggtcc caagaattag gattaattct caaatgacct
     2341 gcaaaacaaa gaaaacaaac cagcaatagg agacggcgca ccttttcaaa cacaccgtgc
     2401 aaatctgctc tcagacgctt tgacaatact attgcactgc tagctgtcca atacagtagt
     2461 gcaatgaagt cagagcacct gttaacagca cttgaggacc ggccacgctg atgaccctac
     2521 ctttgagaac aatccctggc caatgccctt ttaacattgc actgctttaa taactgggtt
     2581 atcagtagta caatataaaa ggggcactgg acagacagta cacaatcagg gtgctccttt
     2641 tgcagccaac tgctgttact ctcgcccaga aatctgcgcc aacatggaca cacccagtgg
     2701 agcaagccat ctgattttca tggtcagatt gtaaagaagc aattactctt ggacagttta
     2761 agcagaaaag ccaccaagtc aagtcactgg tcatatgatg aactcgaaga tctaataaaa
     2821 gttaatttaa gcgttcatta caataaattc aagtgaatct gttttaagct agcctttaac
     2881 gtttttaaat tccttcaaac aaattagttc ctctttgcag ttcctcattt ctcggtgagc
     2941 tggtgcttta atccctttac tttttcttcc tctagggagt gatggttggc atgggacgaa
     3001 ttaattcctg cagcccgggg gtaccggtcg ccaccatgaa caccccggga attaacctga
     3061 tcaaggagga catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc cacgccttcg
     3121 tgatcgaggg cgagggcaag ggcaagccct acgagggcac ccagaccgcc aacctgaccg
     3181 tgaaggaggg cgcccccctg cccttcagct acgacatcct gaccaccgcc gtgcactacg
     3241 gcaaccgggt gttcaccaag taccccgagg acatccccga ctacttcaag cagagcttcc
     3301 ccgagggcta cagctgggag cgcaccatga ccttcgagga caagggcatc tgcaccatcc
     3361 gcagcgacat cagcctggag ggcgactgct tcttccagaa cgtgcgcttc aagggcacca
     3421 acttcccccc caacggcccc gtgatgcaga agaaaaccct gaagtgggag cccagcaccg
     3481 agaagctgca cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc ctgctgctgg
     3541 agggcggcgg ccactacctg tgcgacttca agaccaccta caaggccaag aaggtggtgc
     3601 agctgcccga cgcccacttc gtggaccacc gcatcgagat cctgggcaac gacagcgact
     3661 acaacaaggt gaagctgtac gagcacgccg tggcccgcta cagccccctg cccagccagg
     3721 tgtggtaatc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3781 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     3841 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3901 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3961 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4021 gctcattttt taaccaatag gccgaaatcg gcggatcctg cggccgcaag ggaaaataga
     4081 atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa
     4141 aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt
     4201 gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat
     4261 tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc
     4321 cactagatct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     4381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4621 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4681 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4741 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4801 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4861 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4921 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4981 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5041 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5101 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5161 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5221 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5281 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5341 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5401 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5461 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5521 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5581 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5641 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5701 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5761 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5821 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5881 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5941 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6001 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6061 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6121 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6181 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6241 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6301 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6361 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6421 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6481 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     6541 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6601 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6661 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6721 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6781 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6841 cttgacgggg aaagccggcc acaatgacgg ctactacatg gtgccattcc ttcctcttta
     6901 taggaatgga gactacctcc tgtccaacaa tgctcttgga tacgagtacg cctacctgtt
     6961 ggacccaggt cattgcacaa caccagaaat gccctctgat ctgcaaaaga cgtgaatatc
     7021 tgttcagaca cccatatcca ctctgttcca cacaggtcag aggtttgtcc aggagttctt
     7081 gacagaggtg taaaaagtac tcaaaaattt tactcaagtg aaagtacaag tacttaggga
     7141 aaattttact caattaaaag taaaagtatc tggctagaat cttacttgag taaaagtaaa
     7201 aaagtactcc attaaaattg tacttgagta ttaaggaagt aaaaggctag caagatctgc
     7261 tcgagataca tttgattaga agagtttgac ttgggttgat tatagcaaat agtggaacac
     7321 aaacagattt tgaaatattc ttgcaaattt atttattaaa tccattcaaa tattgtcatt
     7381 ttttgtactg tgtaatttat attgatttgc tcatacaata tttacatttc gcttaaacaa
     7441 tacactatta tttatattta aaaatagcta tatataaatc aaatgtatag ctctttttta
     7501 cacaattaat caaagctaaa atatttgtca cacaaataca gtaagccact tcacaatgat
     7561 atcttcaaaa tctgtgcttg actgaatcat ttcatttatt gttttgtaat tcacattata
     7621 ccttacgttc tccaggcaca gtttgaattg ttgataaatg aagataatac atctgattta
     7681 attagtctcc tttgtttata ataactttaa tctaatttga tagtaataat tccagcaata
     7741 catccattac aataaaaatc gcttttttat tttgcacatt agtttgccgg tatttgttta
     7801 aagtttcacc atacttttaa atcatgtgct atttgaaaag ggctgttatg actggaatta
     7861 aatgtttttt tttatatcag tgttttagat tgaagtacaa tatttatagg taaaattcac
     7921 cagcatatgc ttaaagata
//
