LOCUS       sequence-182664-        7847 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 7847)
  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 7847)
  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 216a-Dendra2
FEATURES             Location/Qualifiers
     source          1..7847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1706..1715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2929..3621
                     /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    3732..3853
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3769..3788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3823..3842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4231..4251)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4231..4249)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4270..4286)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4270..4286)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4283..4305)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4294..4310
                     /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(4318..4324,4325..4342,4343..4348))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4363..4384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4501..4518)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4672..5260)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4752..4771)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5431..6222,6223..6291))
                     /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     6054..6073
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6292..6396)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6559..6580)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttgtgtcct tctaaggtct gtttgtggtt taagctgttt gttgacacac gcttgtcatc
       61 tccatttttc tgtgaagtta ttgtcagttt attgcacact gggttgcatt aagctgcagt
      121 tttttcacaa cagttttcca cattgtaaca gtcagtctcc aaattattcc atcagtcctc
      181 aaatttagtc atttgaggcc atgaaaacac ttaaaactac atgaaggtag tttggcatta
      241 ggttttagac ttgttcttgt ttggcatatt gcaacctgtg taaaaatgat ttgattttta
      301 tttttttttt ttttttttca gaaagaaacg gtccaaaaag agttatgtga acatttatcc
      361 atgtgtttaa tgttttttaa attaaaaaaa aacattttta ttaaaccact tgtgactaaa
      421 ccttttttag ttattctatt ttgtgacgag tctgaatggc caaagtactc ttgaatccaa
      481 aatctttata gtaaaatgtt taatcttact tgcagtgttg ggtataattt gtaacaaagt
      541 tgaattccat atctgctgag tgaaaaagta aagtaagaga ttgctgtaaa tatttaggtt
      601 atttatttat taatacaata tagaaagtaa caaaacacaa tgtaaaaaat caataaagaa
      661 agtaaatatt aaaagtaatg ataaactaat gatgtaaaca atattagcaa caaagtttgt
      721 acaattcagg gagggtaaag aatttcacct aatagtcaaa ctctgtatta aataatataa
      781 aatctttgtt cttagtctga agaggagact gtgagagaaa gcttactggt tgttaaatta
      841 gaaaatttgt aacttgcaga ttaaaatgat ttaaataatt ttataaaggc catgtttgaa
      901 acaaatgtaa aaacataata aatgttaaga atttcagata ttgagatttt acaatttaaa
      961 aaaagcaata aagtaatagc agagacctac actcttaaag taaattaccc aactctgctt
     1021 actagtaact tttggaatag ttactagtat ccattgaaca aatactagaa tctaattaaa
     1081 ttgtgtgagt gtccaatgaa aagattcttg tatttaaaac agtaccagta tctatatatg
     1141 tacaaaattt agtgctagca aaatgcaaat aaatactact gcatataatg gattgcattg
     1201 taaatcgact cgccataata catttatttc ctatttttac ttttaacaaa aaataagttc
     1261 actgagtatt tcatgtcatt aaatcgcatt aaacctcttg catgctttat gtacactagt
     1321 gcagttcgct aaatgaccac cggtgtcact aataacacga tcgtttctga attctgcatt
     1381 aaacgccatt tatacgttgc tcttattagt aaacaatttc tccatcatga gctcgaccag
     1441 tattgcatat ttcatcctct taaatccaga gttgtttctt ttaatcactt tctcttcatg
     1501 ataaaagcag tttttggttt tcagaagttt acaacaaacc cagtcagtta ttgcctcagc
     1561 aaacactgga gcaactgcac acttgtgaaa agcttcttgc gaaacacaaa cctttctctg
     1621 gacttataaa taaaggcatt cagcagctcc accatacacc tccactggtc ttgataaagc
     1681 agatatcagc agtgatacag gtaccgccac catggatgag gaaatcgctg ccctggtcgt
     1741 tgacaacggc tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt
     1801 tttcccctcc attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa
     1861 catgttaatc atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac
     1921 cgatttatat tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg
     1981 cttgttttga gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt
     2041 tcgaagttct tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg
     2101 atacagtata agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt
     2161 ttttcagtgg tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt
     2221 atcgtcagcg tgatgggcaa aaaacagctg gcagtacggg cttaaacatc tgtacgcccc
     2281 ctgctggagt aacctgctat tgagcagctg atttttggca taatctcagc tggcaactgt
     2341 gagtagtgtt ttcatccctc tcacaggcgc tgctggggtt ctgtcacaca cagcacctgc
     2401 aaaacaagac aaaatatcat caaagagaag agggaaacct gtggaaaaaa gttaaactgt
     2461 ggtatggaat gtttataatt tattagttca cccacttcag tttctctggc gctgctggaa
     2521 gcctgagctg catgagagcc aactgctgtt actctcgccc agaaatctgc gccaacatgg
     2581 acacacccag tggagcaagc catctgattt tcatggtcag attgtaaaga agcaattact
     2641 cttggacagt ttaagcagaa aagccaccaa gtcaagtcac tggtcatatg atgaactcga
     2701 agatctaata aaagttaatt taagcgttca ttacaataaa ttcaagtgaa tctgttttaa
     2761 gctagccttt aacgttttta aattccttca aacaaattag ttcctctttg cagttcctca
     2821 tttctcggtg agctggtgct ttaatccctt tactttttct tcctctaggg agtgatggtt
     2881 ggcatgggac gaattaattc ctgcagcccg ggggtaccgg tcgccaccat gaacaccccg
     2941 ggaattaacc tgatcaagga ggacatgcgc gtgaaggtgc acatggaggg caacgtgaac
     3001 ggccacgcct tcgtgatcga gggcgagggc aagggcaagc cctacgaggg cacccagacc
     3061 gccaacctga ccgtgaagga gggcgccccc ctgcccttca gctacgacat cctgaccacc
     3121 gccgtgcact acggcaaccg ggtgttcacc aagtaccccg aggacatccc cgactacttc
     3181 aagcagagct tccccgaggg ctacagctgg gagcgcacca tgaccttcga ggacaagggc
     3241 atctgcacca tccgcagcga catcagcctg gagggcgact gcttcttcca gaacgtgcgc
     3301 ttcaagggca ccaacttccc ccccaacggc cccgtgatgc agaagaaaac cctgaagtgg
     3361 gagcccagca ccgagaagct gcacgtgcgc gacggcctgc tggtgggcaa catcaacatg
     3421 gccctgctgc tggagggcgg cggccactac ctgtgcgact tcaagaccac ctacaaggcc
     3481 aagaaggtgg tgcagctgcc cgacgcccac ttcgtggacc accgcatcga gatcctgggc
     3541 aacgacagcg actacaacaa ggtgaagctg tacgagcacg ccgtggcccg ctacagcccc
     3601 ctgcccagcc aggtgtggta atctagatca taatcagcca taccacattt gtagaggttt
     3661 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3721 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3781 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3841 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3901 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcggatc ctgcggccgc
     3961 aagggaaaat agaatgaagt gatctccaaa aaataagtac tttttgactg taaataaaat
     4021 tgtaaggagt aaaaagtact tttttttcta aaaaaatgta attaagtaaa agtaaaagta
     4081 ttgattttta attgtactca agtaaagtaa aaatccccaa aaataatact taagtacagt
     4141 aatcaagtaa aattactcaa gtactttaca cctctggttc ttgaccccct accttcagca
     4201 agcccagcag atccactaga tcttttgttc cctttagtga gggttaattg cgcgcttggc
     4261 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4321 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4381 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4441 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4501 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4561 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4621 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4681 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4741 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4801 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4861 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4921 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4981 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5041 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5101 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5161 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5221 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5281 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5341 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5401 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5461 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5521 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5581 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5641 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5701 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5761 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5821 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5881 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5941 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6001 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6061 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6121 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6181 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6241 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6301 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6361 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6421 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     6481 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     6541 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     6601 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     6661 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     6721 ccccgattta gagcttgacg gggaaagccg gccacaatga cggctactac atggtgccat
     6781 tccttcctct ttataggaat ggagactacc tcctgtccaa caatgctctt ggatacgagt
     6841 acgcctacct gttggaccca ggtcattgca caacaccaga aatgccctct gatctgcaaa
     6901 agacgtgaat atctgttcag acacccatat ccactctgtt ccacacaggt cagaggtttg
     6961 tccaggagtt cttgacagag gtgtaaaaag tactcaaaaa ttttactcaa gtgaaagtac
     7021 aagtacttag ggaaaatttt actcaattaa aagtaaaagt atctggctag aatcttactt
     7081 gagtaaaagt aaaaaagtac tccattaaaa ttgtacttga gtattaagga agtaaaaggc
     7141 tagcaagatc tgctcgagat acatttgatt agaagagttt gacttgggtt gattatagca
     7201 aatagtggaa cacaaacaga ttttgaaata ttcttgcaaa tttatttatt aaatccattc
     7261 aaatattgtc attttttgta ctgtgtaatt tatattgatt tgctcataca atatttacat
     7321 ttcgcttaaa caatacacta ttatttatat ttaaaaatag ctatatataa atcaaatgta
     7381 tagctctttt ttacacaatt aatcaaagct aaaatatttg tcacacaaat acagtaagcc
     7441 acttcacaat gatatcttca aaatctgtgc ttgactgaat catttcattt attgttttgt
     7501 aattcacatt ataccttacg ttctccaggc acagtttgaa ttgttgataa atgaagataa
     7561 tacatctgat ttaattagtc tcctttgttt ataataactt taatctaatt tgatagtaat
     7621 aattccagca atacatccat tacaataaaa atcgcttttt tattttgcac attagtttgc
     7681 cggtatttgt ttaaagtttc accatacttt taaatcatgt gctatttgaa aagggctgtt
     7741 atgactggaa ttaaatgttt ttttttatat cagtgtttta gattgaagta caatatttat
     7801 aggtaaaatt caccagcata tgcttaaaga tagccatgct ccaggtc
//
