LOCUS       sequence-328093-        4357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to sense miRNA activity using fluorescence based tools. 
            hsa-miR-182-5p target in 3' UTR of of mKate. miRNA activity can be 
            measured by the repression of mKate2 relative to EBFP2 fluorescent 
            proteins . Plasmid is inherently unstable; please see Depositor 
            Comments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4357)
  AUTHORS   Gam JJ, Babb J, Weiss R
  TITLE     A mixed antagonistic/synergistic miRNA repression model enables 
            accurate predictions of multi-input miRNA sensor activity.
  JOURNAL   Nat Commun. 2018 Jun 22;9(1):2430. doi: 10.1038/s41467-018-04575-0.
  PUBMED    29934631
REFERENCE   2  (bases 1 to 4357)
  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: "Nat 
            Commun."; date: "2018-06-22"; volume: "9(1):2430. doi"; pages: " 
            10.1038/s41467-018-04575-0"
COMMENT     Sequence Label: LSB-hsa-miR-182-5p
FEATURES             Location/Qualifiers
     source          1..4357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(36..624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(116..135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(795..1586,1587..1655))
                     /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     1418..1437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1656..1727)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    1744..1781
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1812..2411
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1812..1831)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2308..2328
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2512..2531)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2577..2632
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2631..2650)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2993..3009)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2993..3009)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3006..3028)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3017..3033
                     /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(3041..3047,3048..3065,3066..3071))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3086..3107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3321..3340)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3332..4141
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3399..3418)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
       61 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      241 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      361 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      421 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
      481 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      541 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      601 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      661 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      721 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
      781 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      841 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
      901 ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat
      961 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1021 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1081 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     1141 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1201 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1261 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1321 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1381 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     1441 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1501 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1561 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     1621 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     1681 agcatttatc aaggttattg tctcatgagc ggatacatat ttgaatgtac cgaatttctc
     1741 gagggcttgt cgacgacggc ggtctccgtc gtcaggatca tgaattcgac cgcctgtctc
     1801 aaggtgccac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     1861 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     1921 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     1981 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     2041 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     2101 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     2161 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     2221 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     2281 ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     2341 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     2401 ccggtgcctg agctttgaga ctacgcgtga attcactcct caggtgcagg ctgcctatca
     2461 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     2521 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     2581 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     2641 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     2701 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     2761 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     2821 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     2881 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     2941 tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc
     3001 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3061 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3121 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatcgacagt actaagcttg
     3181 gtctaatacc caatctctcg tcttatccag atgttttata cgcctctggc ccgtgtctca
     3241 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     3301 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     3361 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     3421 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     3481 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     3541 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     3601 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     3661 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     3721 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     3781 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     3841 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     3901 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     3961 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     4021 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     4081 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     4141 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     4201 cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga
     4261 ggtatcacta gtattacaga ggtaagttat aacagtcgcc taacccaatt gaggggataa
     4321 cgcaggaaag aacatgtgag caaaaggcca gcaaaag
//
