LOCUS       sequence-459284-        4357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to sense miRNA activity using fluorescence based tools. 
            hsa-miR-338-3p 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 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-338-3p
FEATURES             Location/Qualifiers
     source          1..4357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(841..1650)
                     /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     1564..1583
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1642..1661
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1875..1896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1911..1916,1917..1934,1935..1941)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1949..1965
                     /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     1954..1976
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1973..1989
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1973..1989
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2332..2351
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2350..2405
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     2451..2470
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2571..3170)
                     /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(2654..2674)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3151..3170
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    3201..3238
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        3255..3326
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3327..3395,3396..4187)
                     /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(3545..3564)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct caattgggtt aggcgactgt tataacttac ctctgtaata ctagtgatac
      721 ctctgacgct cagtggaacg acgcgtaact cacgttaagg gattttggtc atgagcttgc
      781 gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga
      841 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
      901 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      961 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1021 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1081 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1141 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1201 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1261 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1321 ttcttctaat acctggaatg ctgtttttcc ggggatcgca gtggtgagta accatgcatc
     1381 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1441 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1501 caactctggc gcatcgggct tcccatacaa gcgatagatt gtcgcacctg attgcccgac
     1561 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1621 cctcgacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     1681 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     1741 ttgagacacg ggccagaggc gtataaaaca tctggataag acgagagatt gggtattaga
     1801 ccaagcttag tactgtcgat ccgctggcac gacaggtttc ccgactggaa agcgggcagt
     1861 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     1921 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     1981 agctatgacc atgattacgc caagcttggg ctgcaggtcg agggatctcc ataagagaag
     2041 agggacagct atgactggga gtagtcagga gaggaggaaa aatctggcta gtaaaacatg
     2101 taaggaaaat tttagggatg ttaaagaaaa aaataacaca aaacaaaata taaaaaaaat
     2161 ctaacctcaa gtcaaggctt ttctatggaa taaggaatgg acagcagggg gctgtttcat
     2221 atactgatga cctctttata gccaaccttt gttcatggca gccagcatat gggcatatgt
     2281 tgccaaactc taaaccaaat actcattctg atgttttaaa tgatttgccc tcccatatgt
     2341 ccttccgagt gagagacaca aaaaattcca acacactatt gcaatgaaaa taaatttcct
     2401 ttattagcca gaagtcagat gctcaagggg cttcatgatg tccccataat ttttggcaga
     2461 gggaaaaaga tctcagtggt atttgtgagc cagggcattg gccacaccag ccaccacctt
     2521 ctgataggca gcctgcacct gaggagtgaa ttcacgcgta gtctcaaagc tcaggcaccg
     2581 ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg
     2641 gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggatgtctc caggaaggcg
     2701 ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc
     2761 ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg ctccttgggc
     2821 cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac
     2881 tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg
     2941 gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg
     3001 acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccgggtcg
     3061 acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggcccgg
     3121 gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat ggtggcacct
     3181 tgagacaggc ggtcgaattc atgatcctga cgacggagac cgccgtcgtc gacaagccct
     3241 cgagaaattc ggtacattca aatatgtatc cgctcatgag acaataacct tgataaatgc
     3301 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3361 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3421 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3481 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3541 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3601 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3661 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3721 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3781 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3841 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3901 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3961 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4021 aatctggagc cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta
     4081 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4141 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4201 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4261 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4321 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
