LOCUS       sequence-182576-        7861 bp    DNA     circular SYN 15-APR-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 7861)
  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 7861)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 22b-Dendra2
FEATURES             Location/Qualifiers
     source          1..7861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1707..1716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2944..3636
                     /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    3747..3868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3784..3803)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3838..3857
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4246..4266)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4246..4264)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4285..4301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4285..4301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4298..4320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4309..4325
                     /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(4333..4339,4340..4357,4358..4363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4378..4399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4516..4533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4687..5275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4767..4786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5446..6237,6238..6306))
                     /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     6069..6088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6307..6411)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6574..6595)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccttgtgtcc ttctaaggtc tgtttgtggt ttaagctgtt tgttgacaca cgcttgtcat
       61 ctccattttt ctgtgaagtt attgtcagtt tattgcacac tgggttgcat taagctgcag
      121 ttttttcaca acagttttcc acattgtaac agtcagtctc caaattattc catcagtcct
      181 caaatttagt catttgaggc catgaaaaca cttaaaacta catgaaggta gtttggcatt
      241 aggttttaga cttgttcttg tttggcatat tgcaacctgt gtaaaaatga tttgattttt
      301 attttttttt tttttttttc agaaagaaac ggtccaaaaa gagttatgtg aacatttatc
      361 catgtgttta atgtttttta aattaaaaaa aaacattttt attaaaccac ttgtgactaa
      421 acctttttta gttattctat tttgtgacga gtctgaatgg ccaaagtact cttgaatcca
      481 aaatctttat agtaaaatgt ttaatcttac ttgcagtgtt gggtataatt tgtaacaaag
      541 ttgaattcca tatctgctga gtgaaaaagt aaagtaagag attgctgtaa atatttaggt
      601 tatttattta ttaatacaat atagaaagta acaaaacaca atgtaaaaaa tcaataaaga
      661 aagtaaatat taaaagtaat gataaactaa tgatgtaaac aatattagca acaaagtttg
      721 tacaattcag ggagggtaaa gaatttcacc taatagtcaa actctgtatt aaataatata
      781 aaatctttgt tcttagtctg aagaggagac tgtgagagaa agcttactgg ttgttaaatt
      841 agaaaatttg taacttgcag attaaaatga tttaaataat tttataaagg ccatgtttga
      901 aacaaatgta aaaacataat aaatgttaag aatttcagat attgagattt tacaatttaa
      961 aaaaagcaat aaagtaatag cagagaccta cactcttaaa gtaaattacc caactctgct
     1021 tactagtaac ttttggaata gttactagta tccattgaac aaatactaga atctaattaa
     1081 attgtgtgag tgtccaatga aaagattctt gtatttaaaa cagtaccagt atctatatat
     1141 gtacaaaatt tagtgctagc aaaatgcaaa taaatactac tgcatataat ggattgcatt
     1201 gtaaatcgac tcgccataat acatttattt cctattttta cttttaacaa aaaataagtt
     1261 cactgagtat ttcatgtcat taaatcgcat taaacctctt gcatgcttta tgtacactag
     1321 tgcagttcgc taaatgacca ccggtgtcac taataacacg atcgtttctg aattctgcat
     1381 taaacgccat ttatacgttg ctcttattag taaacaattt ctccatcatg agctcgacca
     1441 gtattgcata tttcatcctc ttaaatccag agttgtttct tttaatcact ttctcttcat
     1501 gataaaagca gtttttggtt ttcagaagtt tacaacaaac ccagtcagtt attgcctcag
     1561 caaacactgg agcaactgca cacttgtgaa aagcttcttg cgaaacacaa acctttctct
     1621 ggacttataa ataaaggcat tcagcagctc caccatacac ctccactggt cttgataaag
     1681 cagatatcag cagtgataca ggtaccgcca ccatggatga ggaaatcgct gccctggtcg
     1741 ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg agatgatgcc cctcgtgctg
     1801 ttttcccctc cattgttgga cgacccagac atcaggtaca tttaataaac catcctaata
     1861 acatgttaat catattgaac cacattaaac tctcatttgt gcattttaca tcttttaaaa
     1921 ccgatttata ttcgttttta gataatgaag tttattaaag tgttaattca cagctttatt
     1981 gcttgttttg agaatctgct tgcataagtg atttttaaac ttgatctgat tttcgtttgt
     2041 ttcgaagttc ttcagctttg aaagtcagat cttcaattca cagccatgcc atagttttag
     2101 gatacagtat aagtagacga attgggatgc atcgtagatt ttatgctgtg tttgttatgg
     2161 tttttcagtg gtagagattt tcagagtatt attagggtta tagtcagtgt caggtttatt
     2221 tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg gctaggtgtt ggtgctgcca
     2281 tctgtattat tttagcggca ctgctctcaa atatcattct ggagcactgt ttgatgaagc
     2341 attcagattt attttagacc atgtggatta caatgttttg aaacatggta aatgtcttgt
     2401 cggtattgtc tggctggcgt tacttcacag tcgttcttca ctggctagct ttatgtggca
     2461 gcaccttaaa gctgccagtt gaagagctgt tgtgggtaac cttaacaaca gaccacccaa
     2521 agggtcgtgg tcagcaaggt gggattatgc tagccaactg ctgttactct cgcccagaaa
     2581 tctgcgccaa catggacaca cccagtggag caagccatct gattttcatg gtcagattgt
     2641 aaagaagcaa ttactcttgg acagtttaag cagaaaagcc accaagtcaa gtcactggtc
     2701 atatgatgaa ctcgaagatc taataaaagt taatttaagc gttcattaca ataaattcaa
     2761 gtgaatctgt tttaagctag cctttaacgt ttttaaattc cttcaaacaa attagttcct
     2821 ctttgcagtt cctcatttct cggtgagctg gtgctttaat ccctttactt tttcttcctc
     2881 tagggagtga tggttggcat gggacgaatt aattcctgca gcccgggggt accggtcgcc
     2941 accatgaaca ccccgggaat taacctgatc aaggaggaca tgcgcgtgaa ggtgcacatg
     3001 gagggcaacg tgaacggcca cgccttcgtg atcgagggcg agggcaaggg caagccctac
     3061 gagggcaccc agaccgccaa cctgaccgtg aaggagggcg cccccctgcc cttcagctac
     3121 gacatcctga ccaccgccgt gcactacggc aaccgggtgt tcaccaagta ccccgaggac
     3181 atccccgact acttcaagca gagcttcccc gagggctaca gctgggagcg caccatgacc
     3241 ttcgaggaca agggcatctg caccatccgc agcgacatca gcctggaggg cgactgcttc
     3301 ttccagaacg tgcgcttcaa gggcaccaac ttccccccca acggccccgt gatgcagaag
     3361 aaaaccctga agtgggagcc cagcaccgag aagctgcacg tgcgcgacgg cctgctggtg
     3421 ggcaacatca acatggccct gctgctggag ggcggcggcc actacctgtg cgacttcaag
     3481 accacctaca aggccaagaa ggtggtgcag ctgcccgacg cccacttcgt ggaccaccgc
     3541 atcgagatcc tgggcaacga cagcgactac aacaaggtga agctgtacga gcacgccgtg
     3601 gcccgctaca gccccctgcc cagccaggtg tggtaatcta gatcataatc agccatacca
     3661 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3721 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3781 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3841 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta
     3901 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3961 ggatcctgcg gccgcaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt
     4021 gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa
     4081 gtaaaagtaa aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata
     4141 atacttaagt acagtaatca agtaaaatta ctcaagtact ttacacctct ggttcttgac
     4201 cccctacctt cagcaagccc agcagatcca ctagatcttt tgttcccttt agtgagggtt
     4261 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4321 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4381 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4501 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4561 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4621 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4681 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4741 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4801 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4861 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4921 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4981 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5041 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5101 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5161 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5221 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5281 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5341 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5401 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5461 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5521 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5581 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5641 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5701 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5761 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5821 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5881 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5941 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6001 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6061 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6121 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6181 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6241 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6301 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6361 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6421 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     6481 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     6541 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     6601 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     6661 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     6721 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggccac aatgacggct
     6781 actacatggt gccattcctt cctctttata ggaatggaga ctacctcctg tccaacaatg
     6841 ctcttggata cgagtacgcc tacctgttgg acccaggtca ttgcacaaca ccagaaatgc
     6901 cctctgatct gcaaaagacg tgaatatctg ttcagacacc catatccact ctgttccaca
     6961 caggtcagag gtttgtccag gagttcttga cagaggtgta aaaagtactc aaaaatttta
     7021 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
     7081 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
     7141 aaggaagtaa aaggctagca agatctgctc gagatacatt tgattagaag agtttgactt
     7201 gggttgatta tagcaaatag tggaacacaa acagattttg aaatattctt gcaaatttat
     7261 ttattaaatc cattcaaata ttgtcatttt ttgtactgtg taatttatat tgatttgctc
     7321 atacaatatt tacatttcgc ttaaacaata cactattatt tatatttaaa aatagctata
     7381 tataaatcaa atgtatagct cttttttaca caattaatca aagctaaaat atttgtcaca
     7441 caaatacagt aagccacttc acaatgatat cttcaaaatc tgtgcttgac tgaatcattt
     7501 catttattgt tttgtaattc acattatacc ttacgttctc caggcacagt ttgaattgtt
     7561 gataaatgaa gataatacat ctgatttaat tagtctcctt tgtttataat aactttaatc
     7621 taatttgata gtaataattc cagcaataca tccattacaa taaaaatcgc ttttttattt
     7681 tgcacattag tttgccggta tttgtttaaa gtttcaccat acttttaaat catgtgctat
     7741 ttgaaaaggg ctgttatgac tggaattaaa tgtttttttt tatatcagtg ttttagattg
     7801 aagtacaata tttataggta aaattcacca gcatatgctt aaagatagcc atgctccagg
     7861 t
//
