LOCUS       sequence-476200-        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to sense miRNA activity using fluorescence based tools. 
            hsa-miR-92b-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 7277)
  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 7277)
  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-92b-5p
FEATURES             Location/Qualifiers
     source          1..7277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(499..518)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(500..518)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(629..1288)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1203..1222
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1289..1391)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      1492..2080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2332..3141)
                     /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     3055..3074
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3133..3152
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    3366..3387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3402..3407,3408..3425,3426..3432)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3440..3456
                     /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)."
     primer_bind     3445..3467
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3464..3480
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3464..3480
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3823..3842
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    3841..3896
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     3942..3961
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(4062..4661)
                     /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(4145..4165)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4642..4661
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    4692..4729
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        4746..4817
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4818..4886,4887..5678)
                     /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     complement(5036..5055)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5849..6437
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6591..6608
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6867..6885
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6867..6884
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 agaccatgcg atatccgtag gtctcgaaac cgtctcgggg tgaagacatc cgcagtgtac
       61 cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct
      121 caaacaaatg tgtcacaaag taaggattct gacgtctata tcacagacaa aactgtgcta
      181 gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac
      241 tttgcatgtg caaacgcctt caacaacagc attattccag aagatacctt cttccccagc
      301 ccagaaagtt cctgtgatgt caagctggtc gagaaatctt ttgaaacaga tacgaaccta
      361 aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt
      421 aatctgctca tgacgctgcg gctgtggtcc agctgaggaa ttcatcagaa gagcctcgag
      481 gcggccgcat gtgagtctcc ctatagtgag tcgtattaat ttcgcgggcg gaacccctat
      541 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagtagca ccaggcgttt
      601 aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact
      661 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
      721 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
      781 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
      841 agggattggc tgagacaaaa aacatattct caataaaccc tttagggaaa taggccaggt
      901 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
      961 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     1021 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacga aattccggat
     1081 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     1141 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     1201 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     1261 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     1321 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     1381 cgtgccgatc aaagtctcat tttcgccaaa agttgtcatg accaaaatcc cttaacgtga
     1441 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1501 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1561 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1621 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1681 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1741 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1801 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1861 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1921 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1981 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2041 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2101 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2161 tcaattgggt taggcgactg ttataactta cctctgtaat actagtgata cctctgacgc
     2221 tcagtggaac gacgcgtaac tcacgttaag ggattttggt catgagcttg cgccgtcccg
     2281 tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa
     2341 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2401 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2461 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2521 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2581 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2641 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2701 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2761 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2821 tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2881 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2941 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3001 cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg
     3061 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgacgt
     3121 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3181 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     3241 gggccagagg cgtataaaac atctggataa gacgagagat tgggtattag accaagctta
     3301 gtactgtcga tccgctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3361 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3421 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3481 catgattacg ccaagcttgg gctgcaggtc gagggatctc cataagagaa gagggacagc
     3541 tatgactggg agtagtcagg agaggaggaa aaatctggct agtaaaacat gtaaggaaaa
     3601 ttttagggat gttaaagaaa aaaataacac aaaacaaaat ataaaaaaaa tctaacctca
     3661 agtcaaggct tttctatgga ataaggaatg gacagcaggg ggctgtttca tatactgatg
     3721 acctctttat agccaacctt tgttcatggc agccagcata tgggcatatg ttgccaaact
     3781 ctaaaccaaa tactcattct gatgttttaa atgatttgcc ctcccatatg tccttccgag
     3841 tgagagacac aaaaaattcc aacacactat tgcaatgaaa ataaatttcc tttattagcc
     3901 agaagtcaga tgctcaaggg gcttcatgat gtccccataa tttttggcag agggaaaaag
     3961 atctcagtgg tatttgtgag ccagggcatt ggccacacca gccaccacct tctgataggc
     4021 agcctgcacc tgaggagtga attcacgcgt agtctcaaag ctcaggcacc gggcttgcgg
     4081 gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg
     4141 agccgctcgt agaaggggag gttgcggggc gcggatgtct ccaggaaggc gggcaccccg
     4201 gcgcgctcgg ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg
     4261 tggtcgggcg agacgccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc
     4321 gccaggaggc cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg
     4381 cgcgggccga tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc
     4441 gccaccgcgg cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg
     4501 aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc ggtccgggtc gacggtgtgg
     4561 cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc gtacggcccg ggggacgtcg
     4621 tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca tggtggcacc ttgagacagg
     4681 cggtcgaatt catgatcctg acgacggaga ccgccgtcgt cgacaagccc tcgagaaatt
     4741 cggtacattc aaatatgtat ccgctcatga gacaataacc ttgataaatg cttcaataat
     4801 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4861 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4921 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4981 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5041 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5101 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5161 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5221 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5281 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5341 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5401 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5461 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5521 ccggtgagcg tggttctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5581 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5641 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5701 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5761 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5821 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5881 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5941 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6001 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6061 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6121 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6181 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6241 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6301 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6361 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6421 ggcggagcct atggaaaaac gccagcaatg cggccttttt acggttcctg gccttttgct
     6481 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6541 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6601 tgagcgagga agccgaaaaa tcaataatca gacaacaaga tgtgcgaact cgatatttta
     6661 cacgactctc tttaccaatt ctgccccgaa ttacacttaa aacgactcaa cagcttaacg
     6721 ttggcttgcc acgcattact tgactgtaaa actctcactc ttaccgaact tggccgtaac
     6781 ctgccaacca aagcgagaac aaaacataac atcaaacgaa tcgaccgatt gttaggtaat
     6841 cgtcacttgc aggaaggttt aaacgcattt aggtgacact atagaagtgt gtatcgctcg
     6901 aggctcttca gcatctagag ccaccggcat gagccaccat gagcctcggg ctcctgtgct
     6961 gtgcggcctt ttctctcctg tgggcaggtc ccgtgaatgc tggtgtcact cagaccccaa
     7021 aattccacat cttgaaaacg ggacagagca tgacactgca gtgtgcccag gatatgaacc
     7081 atggatacat gtcctggtat cgacaagacc caggcatggg gctgagactg atttactact
     7141 cagctgctgc tggtactact gacaaagaag tccccaatgg ctacaatgtc agcaggttga
     7201 acacagagga tttcccactc aggctggtgt cggctgctcc ctcccagaca agtagtcttc
     7261 gtcacgagac gtaatgg
//
