LOCUS       sequence-182671-        7779 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 7779)
  AUTHORS   Hsu AY, Wang D, Gurol T, Zhou W, Zhu X, Lu HY, Deng Q
  TITLE     Overexpression of microRNA-722 fine-tunes neutrophilic inflammation 
            through inhibiting Rac2 in zebrafish.
  JOURNAL   Dis Model Mech. 2017 Sep 27. pii: dmm.030791. doi: 
            10.1242/dmm.030791.
  PUBMED    28954734
REFERENCE   2  (bases 1 to 7779)
  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: "Dis Model 
            Mech. 2017 Sep 27. pii: dmm.030791. doi: 10.1242/dmm.030791."
COMMENT     Sequence Label: Tol2-lyzC-microRNA 722-Dendra2
FEATURES             Location/Qualifiers
     source          1..7779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1733..1742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2888..3580
                     /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    3691..3812
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3728..3747)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3782..3801
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3936..4123,4124..4135)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4190..4210)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4190..4208)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4229..4245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4229..4245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4242..4264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4253..4269
                     /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(4277..4283,4284..4301,4302..4307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4322..4343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4631..5219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4711..4730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5390..6181,6182..6250))
                     /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     6013..6032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6251..6355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6518..6539)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6935..6946,6947..7084)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tgcttaaaga tagccatgct ccaggtcctt gtgtccttct aaggtctgtt tgtggtttaa
       61 gctgtttgtt gacacacgct tgtcatctcc atttttctgt gaagttattg tcagtttatt
      121 gcacactggg ttgcattaag ctgcagtttt ttcacaacag ttttccacat tgtaacagtc
      181 agtctccaaa ttattccatc agtcctcaaa tttagtcatt tgaggccatg aaaacactta
      241 aaactacatg aaggtagttt ggcattaggt tttagacttg ttcttgtttg gcatattgca
      301 acctgtgtaa aaatgatttg atttttattt tttttttttt tttttcagaa agaaacggtc
      361 caaaaagagt tatgtgaaca tttatccatg tgtttaatgt tttttaaatt aaaaaaaaac
      421 atttttatta aaccacttgt gactaaacct tttttagtta ttctattttg tgacgagtct
      481 gaatggccaa agtactcttg aatccaaaat ctttatagta aaatgtttaa tcttacttgc
      541 agtgttgggt ataatttgta acaaagttga attccatatc tgctgagtga aaaagtaaag
      601 taagagattg ctgtaaatat ttaggttatt tatttattaa tacaatatag aaagtaacaa
      661 aacacaatgt aaaaaatcaa taaagaaagt aaatattaaa agtaatgata aactaatgat
      721 gtaaacaata ttagcaacaa agtttgtaca attcagggag ggtaaagaat ttcacctaat
      781 agtcaaactc tgtattaaat aatataaaat ctttgttctt agtctgaaga ggagactgtg
      841 agagaaagct tactggttgt taaattagaa aatttgtaac ttgcagatta aaatgattta
      901 aataatttta taaaggccat gtttgaaaca aatgtaaaaa cataataaat gttaagaatt
      961 tcagatattg agattttaca atttaaaaaa agcaataaag taatagcaga gacctacact
     1021 cttaaagtaa attacccaac tctgcttact agtaactttt ggaatagtta ctagtatcca
     1081 ttgaacaaat actagaatct aattaaattg tgtgagtgtc caatgaaaag attcttgtat
     1141 ttaaaacagt accagtatct atatatgtac aaaatttagt gctagcaaaa tgcaaataaa
     1201 tactactgca tataatggat tgcattgtaa atcgactcgc cataatacat ttatttccta
     1261 tttttacttt taacaaaaaa taagttcact gagtatttca tgtcattaaa tcgcattaaa
     1321 cctcttgcat gctttatgta cactagtgca gttcgctaaa tgaccaccgg tgtcactaat
     1381 aacacgatcg tttctgaatt ctgcattaaa cgccatttat acgttgctct tattagtaaa
     1441 caatttctcc atcatgagct cgaccagtat tgcatatttc atcctcttaa atccagagtt
     1501 gtttctttta atcactttct cttcatgata aaagcagttt ttggttttca gaagtttaca
     1561 acaaacccag tcagttattg cctcagcaaa cactggagca actgcacact tgtgaaaagc
     1621 ttcttgcgaa acacaaacct ttctctggac ttataaataa aggcattcag cagctccacc
     1681 atacacctcc actggtcttg ataaagcaga tatcagcagt gatacaggta ccgccaccat
     1741 ggatgaggaa atcgctgccc tggtcgttga caacggctcc ggtatgtgca aagccggttt
     1801 tgctggagat gatgcccctc gtgctgtttt cccctccatt gttggacgac ccagacatca
     1861 ggtacattta ataaaccatc ctaataacat gttaatcata ttgaaccaca ttaaactctc
     1921 atttgtgcat tttacatctt ttaaaaccga tttatattcg tttttagata atgaagttta
     1981 ttaaagtgtt aattcacagc tttattgctt gttttgagaa tctgcttgca taagtgattt
     2041 ttaaacttga tctgattttc gtttgtttcg aagttcttca gctttgaaag tcagatcttc
     2101 aattcacagc catgccatag ttttaggata cagtataagt agacgaattg ggatgcatcg
     2161 tagattttat gctgtgtttg ttatggtttt tcagtggtag agattttcag agtattatta
     2221 gggttatagt cagtgtcagg tttatttatc gtcagcgtga tgggcaaaaa acagctggca
     2281 gtacgggcta atcaggactg tgttgctgtc tgtgtggctg cgcagatgtt ggggacgtcc
     2341 agcggagacc tgtgtattgg aacggagtgg aatttgaaac gttttggcca aaaatgtagc
     2401 catggcaaag gggttttttg cagaaacgtt tcagatttcg ctccgttctc cggcttcgga
     2461 aaaaacctgc tgctgccgag aggaagacga agaggagcca actgctgtta ctctcgccca
     2521 gaaatctgcg ccaacatgga cacacccagt ggagcaagcc atctgatttt catggtcaga
     2581 ttgtaaagaa gcaattactc ttggacagtt taagcagaaa agccaccaag tcaagtcact
     2641 ggtcatatga tgaactcgaa gatctaataa aagttaattt aagcgttcat tacaataaat
     2701 tcaagtgaat ctgttttaag ctagccttta acgtttttaa attccttcaa acaaattagt
     2761 tcctctttgc agttcctcat ttctcggtga gctggtgctt taatcccttt actttttctt
     2821 cctctaggga gtgatggttg gcatgggacg aattaattcc tgcagcccgg gggtaccggt
     2881 cgccaccatg aacaccccgg gaattaacct gatcaaggag gacatgcgcg tgaaggtgca
     2941 catggagggc aacgtgaacg gccacgcctt cgtgatcgag ggcgagggca agggcaagcc
     3001 ctacgagggc acccagaccg ccaacctgac cgtgaaggag ggcgcccccc tgcccttcag
     3061 ctacgacatc ctgaccaccg ccgtgcacta cggcaaccgg gtgttcacca agtaccccga
     3121 ggacatcccc gactacttca agcagagctt ccccgagggc tacagctggg agcgcaccat
     3181 gaccttcgag gacaagggca tctgcaccat ccgcagcgac atcagcctgg agggcgactg
     3241 cttcttccag aacgtgcgct tcaagggcac caacttcccc cccaacggcc ccgtgatgca
     3301 gaagaaaacc ctgaagtggg agcccagcac cgagaagctg cacgtgcgcg acggcctgct
     3361 ggtgggcaac atcaacatgg ccctgctgct ggagggcggc ggccactacc tgtgcgactt
     3421 caagaccacc tacaaggcca agaaggtggt gcagctgccc gacgcccact tcgtggacca
     3481 ccgcatcgag atcctgggca acgacagcga ctacaacaag gtgaagctgt acgagcacgc
     3541 cgtggcccgc tacagccccc tgcccagcca ggtgtggtaa tctagatcat aatcagccat
     3601 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     3661 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     3721 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3781 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     3841 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3901 cggcggatcc tgcggccgca agggaaaata gaatgaagtg atctccaaaa aataagtact
     3961 ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa
     4021 ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa
     4081 aataatactt aagtacagta atcaagtaaa attactcaag tactttacac ctctggttct
     4141 tgacccccta ccttcagcaa gcccagcaga tccactagat cttttgttcc ctttagtgag
     4201 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4261 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4321 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4381 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4441 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5041 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5221 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5281 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5341 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5401 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5461 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5521 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5581 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5641 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5701 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5761 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5821 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5881 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5941 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6001 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6061 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6121 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6181 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6241 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6301 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6361 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     6421 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     6481 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     6541 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6601 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6661 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg ccacaatgac
     6721 ggctactaca tggtgccatt ccttcctctt tataggaatg gagactacct cctgtccaac
     6781 aatgctcttg gatacgagta cgcctacctg ttggacccag gtcattgcac aacaccagaa
     6841 atgccctctg atctgcaaaa gacgtgaata tctgttcaga cacccatatc cactctgttc
     6901 cacacaggtc agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat
     6961 tttactcaag tgaaagtaca agtacttagg gaaaatttta ctcaattaaa agtaaaagta
     7021 tctggctaga atcttacttg agtaaaagta aaaaagtact ccattaaaat tgtacttgag
     7081 tattaaggaa gtaaaaggct agcaagatct gctcgagata catttgatta gaagagtttg
     7141 acttgggttg attatagcaa atagtggaac acaaacagat tttgaaatat tcttgcaaat
     7201 ttatttatta aatccattca aatattgtca ttttttgtac tgtgtaattt atattgattt
     7261 gctcatacaa tatttacatt tcgcttaaac aatacactat tatttatatt taaaaatagc
     7321 tatatataaa tcaaatgtat agctcttttt tacacaatta atcaaagcta aaatatttgt
     7381 cacacaaata cagtaagcca cttcacaatg atatcttcaa aatctgtgct tgactgaatc
     7441 atttcattta ttgttttgta attcacatta taccttacgt tctccaggca cagtttgaat
     7501 tgttgataaa tgaagataat acatctgatt taattagtct cctttgttta taataacttt
     7561 aatctaattt gatagtaata attccagcaa tacatccatt acaataaaaa tcgctttttt
     7621 attttgcaca ttagtttgcc ggtatttgtt taaagtttca ccatactttt aaatcatgtg
     7681 ctatttgaaa agggctgtta tgactggaat taaatgtttt tttttatatc agtgttttag
     7741 attgaagtac aatatttata ggtaaaattc accagcata
//
