LOCUS       sequence-182846-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  As a control of miRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  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 7630)
  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-lyzC-RFP-miR-30 backbone
FEATURES             Location/Qualifiers
     source          1..7630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(557..568,569..706)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      3136..3145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4103..4112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4109..4816
                     /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"
     polyA_signal    4943..5064
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4980..4999)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5034..5053
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(5188..5375,5376..5387)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5442..5462)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5442..5460)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5481..5497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5481..5497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5494..5516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5505..5521
                     /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(5529..5535,5536..5553,5554..5559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5574..5595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5712..5729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5883..6471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5963..5982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6642..7433,7434..7502))
                     /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     7265..7284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7503..7607)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggccacaatg acggctacta catggtgcca
      361 ttccttcctc tttataggaa tggagactac ctcctgtcca acaatgctct tggatacgag
      421 tacgcctacc tgttggaccc aggtcattgc acaacaccag aaatgccctc tgatctgcaa
      481 aagacgtgaa tatctgttca gacacccata tccactctgt tccacacagg tcagaggttt
      541 gtccaggagt tcttgacaga ggtgtaaaaa gtactcaaaa attttactca agtgaaagta
      601 caagtactta gggaaaattt tactcaatta aaagtaaaag tatctggcta gaatcttact
      661 tgagtaaaag taaaaaagta ctccattaaa attgtacttg agtattaagg aagtaaaagg
      721 ctagcaagat ctgctcgaga tacatttgat tagaagagtt tgacttgggt tgattatagc
      781 aaatagtgga acacaaacag attttgaaat attcttgcaa atttatttat taaatccatt
      841 caaatattgt cattttttgt actgtgtaat ttatattgat ttgctcatac aatatttaca
      901 tttcgcttaa acaatacact attatttata tttaaaaata gctatatata aatcaaatgt
      961 atagctcttt tttacacaat taatcaaagc taaaatattt gtcacacaaa tacagtaagc
     1021 cacttcacaa tgatatcttc aaaatctgtg cttgactgaa tcatttcatt tattgttttg
     1081 taattcacat tataccttac gttctccagg cacagtttga attgttgata aatgaagata
     1141 atacatctga tttaattagt ctcctttgtt tataataact ttaatctaat ttgatagtaa
     1201 taattccagc aatacatcca ttacaataaa aatcgctttt ttattttgca cattagtttg
     1261 ccggtatttg tttaaagttt caccatactt ttaaatcatg tgctatttga aaagggctgt
     1321 tatgactgga attaaatgtt tttttttata tcagtgtttt agattgaagt acaatattta
     1381 taggtaaaat tcaccagcat atgcttaaag atagccatgc tccaggtcct tgtgtccttc
     1441 taaggtctgt ttgtggttta agctgtttgt tgacacacgc ttgtcttctc catttttctg
     1501 tgaagttatt gtcagtttat tgcacactgg gttgcattaa gctgcagttt tttcacaaca
     1561 gttttccaca ttgtaacagt cagtctccaa attattccat cagtcctcaa atttagtcat
     1621 ttgaggccat gaaaacactt aaaactacat gaaggtagtt tggcattagg ttttagactt
     1681 gttcttgttt ggcatattgc aacctgtgta aaaatgattt gatttttatt tttttttttt
     1741 ttttttcaga aagaaacggt ccaaaaagag ttatgtgaac atttatccat gtgtttaatg
     1801 ttttttaaat taaaaaaaaa catttttatt aaaccacttg tgactaaacc ttttttagtt
     1861 attctatttt gtgacgagtc tgaatggcca aagtactctt gaatccaaaa tctttatagt
     1921 aaaatgttta atcttacttg cagtgttggg tataatttgt aacaaagttg aattccatat
     1981 ctgctgagtg aaaaagtaaa gtaagagatt gctgtaaata tttaggttat ttatttatta
     2041 atacaatata gaaagtaaca aaacacaatg taaaaaatca ataaagaaag taaatattaa
     2101 aagtaatgat aaactaatga tgtaaacaat attagcaaca aagtttgtac aattcaggga
     2161 gggtaaagaa tttcacctaa tagtcaaact ctgtattaaa taatataaaa tctttgttct
     2221 tagtctgaag aggagactgt gagagaaagc ttactggttg ttaaattaga aaatttgtaa
     2281 cttgcagatt aaaatgattt aaataatttt ataaaggcca tgtttgaaac aaatgtaaaa
     2341 acataataaa tgttaagaat ttcagatatt gagattttac aatttaaaaa aagcaataaa
     2401 gtaatagcag agacctacac tcttaaagta aattacccaa ctctgcttac tagtaacttt
     2461 tggaatagtt actagtatcc attgaacaaa tactagaatc taattaaatt gtgtgagtgt
     2521 ccaatgaaaa gattcttgta tttaaaacag taccagtatc tatatatgta caaaatttag
     2581 tgctagcaaa atgcaaataa atactactgc atataatgga ttgcattgta aatcgactcg
     2641 ccataataca tttatttcct atttttactt ttaacaaaaa ataagttcac tgagtatttc
     2701 atgtcattaa atcgcattaa acctcttgca tgctttatgt acactagtgc agttcgctaa
     2761 atgaccaccg gtgtcactaa taacacgatc gtttctgaat tctgcattaa acgccattta
     2821 tacgttgctc ttattagtaa acaatttctc catcatgagc tcgaccagta ttgcatattt
     2881 catcctctta aatccagagt tgtttctttt aatcactttc tcttcatgat aaaagcagtt
     2941 tttggttttc agaagtttac aacaaaccca gtcagttatt gcctcagcaa acactggagc
     3001 aactgcacac ttgtgaaaag cttcttgcga aacacaaacc tttctctgga cttataaata
     3061 aaggcattca gcagctccac catacacctc cactggtctt gataaagcag atatcagcag
     3121 tgatacaggt acctcgccac catggatgag gaaatcgctg ccctggtcgt tgacaacggc
     3181 tccggtatgt gcaaagccgg ttttgctgga gatgatgccc ctcgtgctgt tttcccctcc
     3241 attgttggac gacccagaca tcaggtacat ttaataaacc atcctaataa catgttaatc
     3301 atattgaacc acattaaact ctcatttgtg cattttacat cttttaaaac cgatttatat
     3361 tcgtttttag ataatgaagt ttattaaagt gttaattcac agctttattg cttgttttga
     3421 gaatctgctt gcataagtga tttttaaact tgatctgatt ttcgtttgtt tcgaagttct
     3481 tcagctttga aagtcagatc ttcaattcac agccatgcca tagttttagg atacagtata
     3541 agtagacgaa ttgggatgca tcgtagattt tatgctgtgt ttgttatggt ttttcagtgg
     3601 tagagatttt cagagtatta ttagggttat agtcagtgtc aggtttattt atcgtcagcg
     3661 tgatgggcaa aaaacagctg gcagtacggg ctatgtcttc gtctagaaga agacaaagcc
     3721 aactgctgtt actctcgccc agaaatctgc gccaacatgg acacacccag tggagcaagc
     3781 catctgattt tcatggtcag attgtaaaga agcaattact cttggacagt ttaagcagaa
     3841 aagccaccaa gtcaagtcac tggtcatatg atgaactcga agatctaata aaagttaatt
     3901 taagcgttca ttacaataaa ttcaagtgaa tctgttttaa gctagccttt aacgttttta
     3961 aattccttca aacaaattag ttcctctttg cagttcctca tttctcggtg agctggtgct
     4021 ttaatccctt tactttttct tcctctaggg agtgatggtt ggcatgggac gaattaattc
     4081 ctgcagcccg ggggtaccgg tcgccaccat ggtgtctaag ggcgaagagc tgattaagga
     4141 gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac
     4201 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
     4261 gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcag
     4321 aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct tccctgaggg
     4381 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     4441 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc
     4501 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccaaca ccgagatgct
     4561 gtaccccgct gacggcggcc tggaaggcag aagcgacatg gccctgaagc tcgtgggcgg
     4621 gggccacctg atctgcaact tcaagaccac atacagatcc aagaaacccg ctaagaacct
     4681 caagatgccc ggcgtctact atgtggacca cagactggaa agaatcaagg aggccgacaa
     4741 agagacctac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc tccctagcaa
     4801 actggggcac aaacttaagt cgactgcatg catctagatc ataatcagcc ataccacatt
     4861 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     4921 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     4981 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5041 gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat
     5101 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcggat
     5161 cctgcggccg caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact
     5221 gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa
     5281 aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac
     5341 ttaagtacag taatcaagta aaattactca agtactttac acctctggtt cttgaccccc
     5401 taccttcagc aagcccagca gatccactag atcttttgtt ccctttagtg agggttaatt
     5461 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5521 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5581 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5641 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5701 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5761 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5821 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5881 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5941 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6001 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6061 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6121 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6181 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6241 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6301 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6361 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6421 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6481 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6541 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6601 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6661 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6721 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6781 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6841 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6901 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6961 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7021 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7081 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7141 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7201 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7261 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7321 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7381 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7441 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7501 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7561 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7621 aaagtgccac
//
