LOCUS       sequence-182582-        7946 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 7946)
  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 7946)
  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 31-Dendra2
FEATURES             Location/Qualifiers
     source          1..7946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1334..1343
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2656..3348
                     /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    3459..3580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3496..3515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3550..3569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(3704..3891,3892..3903)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3958..3978)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3958..3976)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3997..4013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3997..4013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4010..4032)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4021..4037
                     /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(4045..4051,4052..4069,4070..4075))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4090..4111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4228..4245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4399..4987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4479..4498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5158..5949,5950..6018))
                     /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     5781..5800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6019..6123)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6286..6307)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(6703..6714,6715..6852)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ttttttaaat taaaaaaaaa catttttatt aaaccacttg tgactaaacc ttttttagtt
       61 attctatttt gtgacgagtc tgaatggcca aagtactctt gaatccaaaa tctttatagt
      121 aaaatgttta atcttacttg cagtgttggg tataatttgt aacaaagttg aattccatat
      181 ctgctgagtg aaaaagtaaa gtaagagatt gctgtaaata tttaggttat ttatttatta
      241 atacaatata gaaagtaaca aaacacaatg taaaaaatca ataaagaaag taaatattaa
      301 aagtaatgat aaactaatga tgtaaacaat attagcaaca aagtttgtac aattcaggga
      361 gggtaaagaa tttcacctaa tagtcaaact ctgtattaaa taatataaaa tctttgttct
      421 tagtctgaag aggagactgt gagagaaagc ttactggttg ttaaattaga aaatttgtaa
      481 cttgcagatt aaaatgattt aaataatttt ataaaggcca tgtttgaaac aaatgtaaaa
      541 acataataaa tgttaagaat ttcagatatt gagattttac aatttaaaaa aagcaataaa
      601 gtaatagcag agacctacac tcttaaagta aattacccaa ctctgcttac tagtaacttt
      661 tggaatagtt actagtatcc attgaacaaa tactagaatc taattaaatt gtgtgagtgt
      721 ccaatgaaaa gattcttgta tttaaaacag taccagtatc tatatatgta caaaatttag
      781 tgctagcaaa atgcaaataa atactactgc atataatgga ttgcattgta aatcgactcg
      841 ccataataca tttatttcct atttttactt ttaacaaaaa ataagttcac tgagtatttc
      901 atgtcattaa atcgcattaa acctcttgca tgctttatgt acactagtgc agttcgctaa
      961 atgaccaccg gtgtcactaa taacacgatc gtttctgaat tctgcattaa acgccattta
     1021 tacgttgctc ttattagtaa acaatttctc catcatgagc tcgaccagta ttgcatattt
     1081 catcctctta aatccagagt tgtttctttt aatcactttc tcttcatgat aaaagcagtt
     1141 tttggttttc agaagtttac aacaaaccca gtcagttatt gcctcagcaa acactggagc
     1201 aactgcacac ttgtgaaaag cttcttgcga aacacaaacc tttctctgga cttataaata
     1261 aaggcattca gcagctccac catacacctc cactggtctt gataaagcag atatcagcag
     1321 tgatacaggt accgccacca tggatgagga aatcgctgcc ctggtcgttg acaacggctc
     1381 cggtatgtgc aaagccggtt ttgctggaga tgatgcccct cgtgctgttt tcccctccat
     1441 tgttggacga cccagacatc aggtacattt aataaaccat cctaataaca tgttaatcat
     1501 attgaaccac attaaactct catttgtgca ttttacatct tttaaaaccg atttatattc
     1561 gtttttagat aatgaagttt attaaagtgt taattcacag ctttattgct tgttttgaga
     1621 atctgcttgc ataagtgatt tttaaacttg atctgatttt cgtttgtttc gaagttcttc
     1681 agctttgaaa gtcagatctt caattcacag ccatgccata gttttaggat acagtataag
     1741 tagacgaatt gggatgcatc gtagatttta tgctgtgttt gttatggttt ttcagtggta
     1801 gagattttca gagtattatt agggttatag tcagtgtcag gtttatttat cgtcagcgtg
     1861 atgggcaaaa aacagctggc agtacgggct aggtttggct ttgaactgtg gaaatgaagc
     1921 tgtttttgtg gagacgtcca tcatttctcc tgcatctgga agagaagaga tggcaagatg
     1981 ttggcatagc tgttaatgtt tatgggcctg ctatgcctcc atattgccat ttctgcactt
     2041 ccaataaaac atcaccaact aaatatgatc aaatgtctgt tatgagctag tttacagttc
     2101 agatgtgcat gttaatgtga aatatatgtc agattgattt acttaaaaat tacactattt
     2161 atttatgtac tgtatgtatt tatttattta ttttactgtt tattcaaatg tattattctg
     2221 tcaaacccgc tattggtttt ccagcagcac gataaaaata ctcagccaac tgctgttact
     2281 ctcgcccaga aatctgcgcc aacatggaca cacccagtgg agcaagccat ctgattttca
     2341 tggtcagatt gtaaagaagc aattactctt ggacagttta agcagaaaag ccaccaagtc
     2401 aagtcactgg tcatatgatg aactcgaaga tctaataaaa gttaatttaa gcgttcatta
     2461 caataaattc aagtgaatct gttttaagct agcctttaac gtttttaaat tccttcaaac
     2521 aaattagttc ctctttgcag ttcctcattt ctcggtgagc tggtgcttta atccctttac
     2581 tttttcttcc tctagggagt gatggttggc atgggacgaa ttaattcctg cagcccgggg
     2641 gtaccggtcg ccaccatgaa caccccggga attaacctga tcaaggagga catgcgcgtg
     2701 aaggtgcaca tggagggcaa cgtgaacggc cacgccttcg tgatcgaggg cgagggcaag
     2761 ggcaagccct acgagggcac ccagaccgcc aacctgaccg tgaaggaggg cgcccccctg
     2821 cccttcagct acgacatcct gaccaccgcc gtgcactacg gcaaccgggt gttcaccaag
     2881 taccccgagg acatccccga ctacttcaag cagagcttcc ccgagggcta cagctgggag
     2941 cgcaccatga ccttcgagga caagggcatc tgcaccatcc gcagcgacat cagcctggag
     3001 ggcgactgct tcttccagaa cgtgcgcttc aagggcacca acttcccccc caacggcccc
     3061 gtgatgcaga agaaaaccct gaagtgggag cccagcaccg agaagctgca cgtgcgcgac
     3121 ggcctgctgg tgggcaacat caacatggcc ctgctgctgg agggcggcgg ccactacctg
     3181 tgcgacttca agaccaccta caaggccaag aaggtggtgc agctgcccga cgcccacttc
     3241 gtggaccacc gcatcgagat cctgggcaac gacagcgact acaacaaggt gaagctgtac
     3301 gagcacgccg tggcccgcta cagccccctg cccagccagg tgtggtaatc tagatcataa
     3361 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3421 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3481 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3541 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     3601 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3661 gccgaaatcg gcggatcctg cggccgcaag ggaaaataga atgaagtgat ctccaaaaaa
     3721 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     3781 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     3841 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     3901 ctggttcttg accccctacc ttcagcaagc ccagcagatc cactagatct tttgttccct
     3961 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4021 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4081 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4141 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4201 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4261 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4321 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4381 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4441 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4501 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4561 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4621 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4681 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4741 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4801 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4861 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4921 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4981 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5041 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5101 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5161 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5221 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5281 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5341 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5401 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5461 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5521 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5581 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5641 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5701 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5761 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5821 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5881 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5941 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6001 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6061 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6121 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     6181 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     6241 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     6301 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6361 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6421 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcc
     6481 acaatgacgg ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc
     6541 tgtccaacaa tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa
     6601 caccagaaat gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca
     6661 ctctgttcca cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac
     6721 tcaaaaattt tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag
     6781 taaaagtatc tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg
     6841 tacttgagta ttaaggaagt aaaaggctag caagatctgc tcgagataca tttgattaga
     6901 agagtttgac ttgggttgat tatagcaaat agtggaacac aaacagattt tgaaatattc
     6961 ttgcaaattt atttattaaa tccattcaaa tattgtcatt ttttgtactg tgtaatttat
     7021 attgatttgc tcatacaata tttacatttc gcttaaacaa tacactatta tttatattta
     7081 aaaatagcta tatataaatc aaatgtatag ctctttttta cacaattaat caaagctaaa
     7141 atatttgtca cacaaataca gtaagccact tcacaatgat atcttcaaaa tctgtgcttg
     7201 actgaatcat ttcatttatt gttttgtaat tcacattata ccttacgttc tccaggcaca
     7261 gtttgaattg ttgataaatg aagataatac atctgattta attagtctcc tttgtttata
     7321 ataactttaa tctaatttga tagtaataat tccagcaata catccattac aataaaaatc
     7381 gcttttttat tttgcacatt agtttgccgg tatttgttta aagtttcacc atacttttaa
     7441 atcatgtgct atttgaaaag ggctgttatg actggaatta aatgtttttt tttatatcag
     7501 tgttttagat tgaagtacaa tatttatagg taaaattcac cagcatatgc ttaaagatag
     7561 ccatgctcca ggtccttgtg tccttctaag gtctgtttgt ggtttaagct gtttgttgac
     7621 acacgcttgt catctccatt tttctgtgaa gttattgtca gtttattgca cactgggttg
     7681 cattaagctg cagttttttc acaacagttt tccacattgt aacagtcagt ctccaaatta
     7741 ttccatcagt cctcaaattt agtcatttga ggccatgaaa acacttaaaa ctacatgaag
     7801 gtagtttggc attaggtttt agacttgttc ttgtttggca tattgcaacc tgtgtaaaaa
     7861 tgatttgatt tttatttttt tttttttttt ttcagaaaga aacggtccaa aaagagttat
     7921 gtgaacattt atccatgtgt ttaatg
//
