LOCUS       sequence-287972-        8869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For miR-223 knock down.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8869)
  AUTHORS   Zhou W, Pal AS, Hsu AY, Gurol T, Zhu X, Wirbisky-Hershberger SE, 
            Freeman JL, Kasinski AL, Deng Q
  TITLE     MicroRNA-223 Suppresses the Canonical NF-kappaB Pathway in Basal 
            Keratinocytes to Dampen Neutrophilic Inflammation.
  JOURNAL   Cell Rep. 2018 Feb 13;22(7):1810-1823. doi: 
            10.1016/j.celrep.2018.01.058.
  PUBMED    29444433
REFERENCE   2  (bases 1 to 8869)
  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"; doi: 
            "10.1016/j.celrep.2018.01"; journalName: "Cell Rep"; date: 
            "2018-02-13- 13"; volume: "22"; issue: "7"; pages: "1810-1823"
COMMENT     Sequence Label: Tol2-krt4-RFP-miR-223 sponge
FEATURES             Location/Qualifiers
     source          1..8869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(32..460)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(142..163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     354..373
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     587..609
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..618
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        628..646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(776..787,788..950)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    1322..1342
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3605..3629
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     3631..3647
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3657..3675
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3713..3722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3719..4426
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKL"
     promoter        complement(4620..4638)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(4667..4691)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4696..4830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4720..4739)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4774..4793
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(6397..6584,6585..6596)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6630..6646)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6630..6646)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(6683..6701)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6722..6738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6722..6738)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6735..6757)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6746..6762
                     /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(6770..6776,6777..6794,6795..6800))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6815..6836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6953..6970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7124..7712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7204..7223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7883..8674,8675..8743))
                     /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     8506..8525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8744..8848)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
       61 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      121 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      181 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      241 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      301 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      361 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      421 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
      481 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      541 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      601 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtaca
      661 ggtcattgca caacaccaga aatgccctct gatctgcaaa agacgtgaat atctgttcag
      721 acacccatat ccactctgtt ccacacaggt cagaggtttg tccaggagtt cttgacagag
      781 gtgtaaaaag tactcaaaaa ttttactcaa gtgaaagtac aagtacttag ggaaaatttt
      841 actcaattaa aagtaaaagt atctggctag aatcttactt gagtaaaagt aaaaaagtac
      901 tccattaaaa ttgtacttga gtattaagga agtaaaagta aaagcaagaa agaaaactag
      961 agattcttgt ttaagctttt aatctcaaaa aacattaaat gaaatgcata caaggtttta
     1021 tcctgcttta gaactgtttg tatttaatta tcaaactata agacagacaa tctaatgcca
     1081 gtacacgcta ctcaaagttg taaaacctca gatttaactt cagtagaagc tgattctcaa
     1141 aattgttagt gtcaagccta gctcttttgg ggctgaaaag caatcctgca gtgctgaaaa
     1201 gcctctcaca ggcagccgat gcgggaagag gtgtattagt cttgatagag aggctgcaaa
     1261 tagcaggaaa cgtgagcaga gactccctgg tgtctgaaac acaggccaga tgggccgata
     1321 tcaactttgt atagaaaagt tggctccgaa ttcgccctta aaactcgagc tcaagcttcg
     1381 aattcctaca gtaaagcttc tccacaatgt cccggagatc cagcccggac atgcattttc
     1441 ttgcctttcc ttcccttcta agtctgacgt ccttttaaga gcttgtgcat gaaagcaaat
     1501 ttagagctta ttactcatct taaacaccca tacaaagtga tgattgccgt accatgatct
     1561 cacacctttc acacctggtt tatactatga tagttgtaga cgattgcgta atgctactaa
     1621 atgcccatca gtgctggctg tgacacccaa ctgctgccat ttcgtgttga cttgcacgag
     1681 aaatgagaaa ttgtctgact atgcagggtg tactatgcgt gggaacttat cagtggtcat
     1741 taaatactat ggtttaaagt tagagcaaag tgtgctgtat ttttgtgtca gcttagctgc
     1801 tgttttgtgt gtaaagtaac aaatgacaaa tactcaaact attgaaatta agtagttttt
     1861 ctcagaaatt gtaatttact aagtagttta aaaatgtgta cttttacttt cccttgagca
     1921 catttttagt gcagtattgg tacttttatt tcactacttt ccttcaacct gcagtcacta
     1981 ctttattttt tcttgtctat gtggattaga caaatcagtc ctgtgattcc tgtccaatca
     2041 aattgcacag aaggtaaatc acatcataat gaacttcctc aagacatggg ccatttataa
     2101 ttgcagcaaa ctgtttgcca gaattaaaag aagatgtcaa aaatctttac acgcattaac
     2161 ccagagactg cttagatgca tgtcactgat gagaagatga cagatgtttt ctgtatgatg
     2221 accgaaataa ccttaaacgc acacaagacg gcacaagacg tcaacatggc tataggttta
     2281 cgttgtaccc cattgcaatg gggacgttgt attttgttta gaaatgaaaa ttaggttgac
     2341 gtcagagctc aacgtcaggt cgatgtcaag gttcaacatc caatctaaaa tcatatatca
     2401 atgtctaatg atgttacagc ttgatgttgt gtggatgtta cccctatgat gtctatcaga
     2461 agttggattg tggttgccat acctaatgaa taaatgtcat tattttacat ttgtttaaga
     2521 tgttggttcg acattggact tatgtcgctt tccaacacaa cctaaatcca ccaaatatta
     2581 acttcctatg acatcgttat tggacatcaa aataacaata tccttagatg ctggctagac
     2641 tttgaattta ggtcaccaca acctatattt aacctaatat taacatctta tgatgttgtg
     2701 tgcctgctgg gcaataacta aatgcactac agaatgttac gtttacacac atgtaaatta
     2761 catgtaaatg cattagcttt ttaagacata atactcacta ctaactactc ttgagtactt
     2821 ttaaaaaagc tactttttac tcatactttg agtaacagtt acaacagata cttttactcg
     2881 cactggattt ttaggcatgt aatggtattt ttactatgat ttttcagtac tctttccact
     2941 actgcagccc tccccataca taatcatatg tttacacata tggtggagtt tagagccata
     3001 aactacatta actttgttag ccgccagcat tactgtgcag aattgtgtgt gtgcacattt
     3061 tccaatatca atacagaagg aaactctgtt ccctgttccc ttgtaaatct caacaatgca
     3121 actgttcagc tcagggggaa aaatgccctg ccagatccaa acgctggcaa aagtggatgg
     3181 aaaaaagcct ttcattaatg taagagttgc tgcgcgcccc acccagataa aaagagcaga
     3241 ggttaacatg ctctctacgg ctgtccagcc aaccagatac tgaggcagaa acacacccgc
     3301 ttgcagatgg tgagagctac actgtctttt tcagagtttc tactggaatg cctgtcctca
     3361 agtctcaagc ctctccttgc attttctcat tccacctggg gcaaagcccc aggctgggtg
     3421 tgacaccatt tatcttacca ctttctctct gtacctgtct aacaggtagg gtgtgtgtga
     3481 gagtgcgtat gtgtgcaagt gtgtgtgtga gagcagtcag ctccaccccc tcaagagtgt
     3541 gtataaaatt ggtcagccag ctgctgagag acacgcagag ggatccttta agggcgaatt
     3601 cgacccaagt ttgtacaaaa aagcaggctt gtaaaacgac ggccagtgag cgcgcgtaat
     3661 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac tcgccaccat
     3721 ggtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     3781 caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     3841 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     3901 cctggctacc agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc
     3961 cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     4021 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     4081 caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac
     4141 actcggctgg gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag
     4201 aagcgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac
     4261 atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca
     4321 cagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc
     4381 tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaagc ggccgctagc
     4441 ggggtatttc tgaactgaca ggaaccttgg ggtattttgt aactgacatc tctcgagggg
     4501 tatttcgtaa ctgacaaatt cgcgggggta tttcttaact gacagcgcta atggggtatt
     4561 ttagaactga cagatcgatc ggggtattta ataactgaca gctagcccag cttttgttcc
     4621 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagcaccc agctttcttg
     4681 tacaaagttg atgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     4741 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     4801 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     4861 caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg
     4921 gccctcgagc atatgagatc tgtcccgatc tttctcttct gtgctgtcaa tttagagtag
     4981 tttttgaggt aatttgggtg cattctctgt aaaaacaaca aataaatatt tatattgtat
     5041 gcaataaatg taacaattac acatacactc tctgattaca gcattaaagg gttagttcrs
     5101 mcmaaaatga rrrsrwgtca ttaatgacat tattttcatt tttgggtgaa ctaacccttt
     5161 aataaaactg acatttgcag ccaattgcac actttataat gttaaatact tgttgatttt
     5221 agaaatacag tagcatttag tgacaatcaa acattaattt tatttctgat tatattgaca
     5281 aaaacacagg actgtaaggc tgacatgaca gggtgaattg atttcattac attagttttg
     5341 atggtaacac ttttacaata aggttcgtta gttaactaca ttagttaaca tgaactaata
     5401 atgaactgca cgtatacatg ttaatttcaa catttactaa tactttatat cttgttaaca
     5461 ttagttaatg cactgtgaac taacatgaac aaacaatgaa catctgtatt tctattaact
     5521 aacgttaaca aagattaaca gatacagtaa caaatgtatt gctcatggtt atttaatgtt
     5581 agttaataca tgaactaatc aacctcattg taaagtgtta ccgttttgac aatacaacat
     5641 gtttggaaaa aaatttctcg cttggttttc ttgtgttttg catgaattca aacacaatga
     5701 gagcacggaa tgataatgat tattcaaagc tgcaataggc gccaaatttc ccgtggacct
     5761 aacgttacca attacaaatg cattttagca ttttattacg tgagccttat attgtcaatc
     5821 gcatgcatta gtgaaaatag gcctactaca catataaaag gcgcgccatc aacgtcaaaa
     5881 catgcacacc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa ctatcagtaa aacaaaatac
     5941 ttaatgtact tacctcaata tgcttcctta ggtttgatgg cgaacttttg aaggccgata
     6001 tttctttatt aagcgggaga cagaggacac atttcatctt gaatgaatct ttatttacac
     6061 cacttaaaga aaagaattcg cgaagatacg gccacgggtg ctcttgatcc tgtggctgat
     6121 tttggactgt gctgctcgca gctgctgatg aatcacatac ttcctccatt ttcttccact
     6181 gattgactgt tataatttcc ctaatttcca ggtcaaggtg ctgtgcattg tggtaataga
     6241 tgtgacatga cgtcacttcc aaaggaccaa tgaacatgtc tgaccaattt catataatgt
     6301 gaaaacgatt ttcataggca gaataaataa catttaaatt aaactgggca tcagcgcaat
     6361 tcaattggtt tggtaatagc aagggaaaat agaatgaagt gatctccaaa aaataagtac
     6421 tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta
     6481 attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa
     6541 aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca cctctggttc
     6601 ttgaccccct accttcagca agcccagcag atccactagt tctagagcgg ccgccaccgc
     6661 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     6721 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6781 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6841 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6901 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6961 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     7021 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     7081 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     7141 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     7201 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7261 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7321 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7381 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7441 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7501 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7561 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7621 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7681 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7741 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7801 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7861 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7921 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7981 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     8041 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     8101 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     8161 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     8221 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     8281 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     8341 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     8401 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     8461 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     8521 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8581 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8641 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8701 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8761 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8821 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcc
//
