LOCUS       sequence-328090-        4357 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used to sense miRNA activity using fluorescence based tools. 
            hsa-miR-124a-3p 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 Apr 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-124a-3p
FEATURES             Location/Qualifiers
     source          1..4357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(413..1204,1205..1273))
                     /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     1036..1055
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1274..1345)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    1362..1399
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1430..2029
                     /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(1430..1449)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1926..1946
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2130..2149)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2195..2250
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2249..2268)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2611..2627)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2611..2627)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2624..2646)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2635..2651
                     /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(2659..2665,2666..2683,2684..2689))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2704..2725
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2939..2958)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2950..3759
                     /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(3017..3036)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(4011..4357,1..242))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4091..4110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
       61 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      121 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      181 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      241 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      301 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      361 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      421 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
      481 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
      541 caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata aaccagccag
      601 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
      661 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      721 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      781 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
      841 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
      901 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
      961 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1021 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1081 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1141 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1201 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1261 gttgaatact catactcttc ctttttcaat attattgaag catttatcaa ggttattgtc
     1321 tcatgagcgg atacatattt gaatgtaccg aatttctcga gggcttgtcg acgacggcgg
     1381 tctccgtcgt caggatcatg aattcgaccg cctgtctcaa ggtgccacca tgaccgagta
     1441 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc
     1501 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga
     1561 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     1621 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     1681 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     1741 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     1801 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     1861 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacatccgc
     1921 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     1981 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag ctttgagact
     2041 acgcgtgaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     2101 aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac
     2161 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     2221 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     2281 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct
     2341 gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     2401 cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt
     2461 gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact agccagattt
     2521 ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg gagatccctc
     2581 gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2641 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2701 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2761 aaacctgtcg tgccagcgga tcgacagtac taagcttggt ctaataccca atctctcgtc
     2821 ttatccagat gttttatacg cctctggccc gtgtctcaaa atctctgatg ttacattgca
     2881 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     2941 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     3001 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     3061 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     3121 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     3181 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     3241 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     3301 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     3361 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     3421 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     3481 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     3541 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     3601 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     3661 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     3721 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     3781 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     3841 cttaacgtga gttacgcgtc gttccactga gcgtcagagg tatcactagt attacagagg
     3901 taagttataa cagtcgccta acccaattga ggggataacg caggaaagaa catgtgagca
     3961 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4021 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4081 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4141 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4201 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4261 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4321 gagtccaacc cggtaagaca cgacttatcg ccactgg
//
