LOCUS       sequence-448314-        8009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of AcrIIC3-mCherry fusion with microRNA target 
            sites in the 3' UTR as a reporter of microRNA repression. BFP 
            expressed separately as a transfection control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8009)
  AUTHORS   Kraus C, Wang J, Zheng H, Broderick J, Ajaykumar N, Zamani M, Yang 
            M, Cecchini K, Liang SQ, Kolumba O, Chase K, Lee J, Xue W, 
            Sontheimer E, Gainetdinov I
  TITLE     Absolute Quantification of Mammalian MicroRNAs for Therapeutic RNA 
            Cleavage and Detargeting.
  JOURNAL   RNA. 2025 May 28:rna.080566.125. doi: 10.1261/rna.080566.125.
  PUBMED    40436630
REFERENCE   2  (bases 1 to 8009)
  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: "RNA. 2025 
            May 28:rna.080566.125. doi: 10.1261/rna.080566.125."
COMMENT     Sequence Label: pEJS1353 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-33b...
FEATURES             Location/Qualifiers
     source          1..8009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             445..468
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             475..495
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             508..1218
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYYAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(653..671)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     921..940
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1311..1328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1317..1428
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1679..2058
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2059..2270
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2267..2291
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2431..2563
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2546..2565
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2608..2626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2632..3342
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    join(3395..3969,3970..3972,3973..3981)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3562..3579)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3789..3808
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4052..4173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4063..4082)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4117..4136
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4308..4326)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4347..4363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4347..4363)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4360..4382)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4371..4387
                     /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(4395..4401,4402..4419,4420..4425))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4440..4461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4578..4595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4749..5337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4829..4848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5508..6299,6300..6368))
                     /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     6131..6150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6369..6473)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6499..6954)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6636..6657)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6848..6867
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7025..8009
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agacggaatt
       61 gtacccgcgg ccgatccacc ggtcgccacc atgttcaaac gggccattat tttcaccagc
      121 ttcaacggct tcgagaaggt cagtcgcacg gaaaaacggc gccttgccaa gataattaac
      181 gccagagtta gtataatcga cgagtatctt cgcgccaaag acaccaacgc ctccttggac
      241 ggacagtata gagcattcct tttcaatgat gagtcaccag cgatgaccga attcctcgct
      301 aagctcaagg cgttcgcaga aagctgtact ggtataagca ttgatgcgtg ggagattgaa
      361 gagagcgagt atgttcgatt gcccgtcgaa cggagagatt tccttgcggc ggccaatggc
      421 aaagaaattt tcaaaattgg cggggattat aaggatgatg atgataaggg ctcccctaag
      481 aagaagagaa aggtgggatc cggaagtatg gtgagcaagg gcgaggagga taacatggcc
      541 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      601 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      661 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      721 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
      781 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
      841 gtgacccagg actcctcact gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
      901 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
      961 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1021 ctgaaggacg gcggccacta ctacgctgag gtcaagacca cctacaaggc caagaagccc
     1081 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1141 gactacacca tcgtggaaca gtacgaacgc gctgagggcc gccactccac cggcggcatg
     1201 gacgagctgt acaagtgata aagcggccat caagctgcaa tgcaacagca atgcacgcaa
     1261 tgcaacagca atgcacgcaa tgcaacagca atgcacagct cgctgatcag cctcgactgt
     1321 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1381 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1441 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1501 agacaatagc ctaggtagat aagtagcatg gcgggtgatc ttcaatattg gccattagcc
     1561 atattattca ttggttatat agcataaatc aatattggct attggccatt gcatacgttg
     1621 tatctatatc ataatatgta catttatatt ggctcatgtc caatatgacc gccatgttgg
     1681 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     1741 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1801 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     1861 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     1921 gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     1981 cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta
     2041 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg
     2101 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     2161 caccaaaatc aacgggactt tccaaaatgt cgtaacaact gcgatcgccc gccccgttga
     2221 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
     2281 accgtcagat cactagaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag
     2341 tcagtgcttc tgacacaaca gtctcgaact taagctgcag tgactctctt aaggtagcct
     2401 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
     2461 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
     2521 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc
     2581 aattacagct cttaaggcta gagtacttaa tacgactcac tataggctag aatggtgtct
     2641 aagggcgaag agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     2701 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     2761 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     2821 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     2881 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     2941 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     3001 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     3061 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     3121 atggccctga agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga
     3181 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     3241 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     3301 agatactgcg acctccctag caaactgggg cacaagctta attgaacgcg tcgagcatgc
     3361 atctagggcg gccaattccg cccctctccc cccccccctc tccctccccc ccccctaacg
     3421 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
     3481 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
     3541 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
     3601 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
     3661 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
     3721 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
     3781 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
     3841 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     3901 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
     3961 gatgataagc ttgccacaac ccgggatcct ctagagtcga cccgggcggc cgcttccctt
     4021 tagtgagggt taatgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     4081 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4141 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4201 tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4261 ggtaaaatcc gataaggatc gatccgggcg gtacccagct tttgttccct ttagtgaggg
     4321 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4381 ctcacaattc cacacaacat acgagccggg agcataaagt gtaaagcctg gggtgcctaa
     4441 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4501 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4561 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4621 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4681 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4741 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4801 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4861 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4921 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4981 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5041 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5101 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5161 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5221 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5281 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5341 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5401 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5461 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5521 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5581 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5641 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5701 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5761 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5821 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5881 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5941 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6001 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6061 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6121 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6181 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6241 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6301 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6361 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6421 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6481 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6541 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6601 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6661 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6721 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6781 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6841 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6901 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     6961 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     7021 gccagtcgac cgccaattca atatggcgta tatggactca tgccaattca atatggtgga
     7081 tctggacctg tgccaattca atatggcgta tatggactcg tgccaattca atatggtgga
     7141 tctggacccc agccaattca atatggcgga cttggcacca tgccaattca atatggcgga
     7201 cctggcactg tgccaactgg ggaggggtct acttggcacg gtgccaagtt tgaggagggg
     7261 tcttggccct gtgccaagtc cgccatattg aattggcatg gtgccaataa tggcggccat
     7321 attggctata tgccaggatc aatatatagg caatatccaa tatggcccta tgccaatatg
     7381 gctattggcc aggttcaata ctatgtattg gccctatgcc atatagtatt ccatatatgg
     7441 gttttcctat tgacgtagat agcccctccc aatgggcggt cccatatacc atatatgggg
     7501 cttcctaata ccgcccatag ccactccccc attgacgtca atggtctcta tatatggtct
     7561 ttcctattga cgtcatatgg gcggtcctat tgacgtatat ggcgcctccc ccattgacgt
     7621 caattacggt aaatggcccg cctggctcaa tgcccattga cgtcaatagg accacccacc
     7681 attgacgtca atgggatggc tcattgccca ttcatatccg ttctcacgcc ccctattgac
     7741 gtcaatgacg gtaaatggcc cacttggcag tacatcaata tctattaata gtaacttggc
     7801 aagtacatta ctattggaag tacgccaggg tacattggca gtactcccat tgacgtcaat
     7861 ggcggtaaat ggcccgcgat ggctgccaag tacatcccca ttgacgtcaa tggggagggg
     7921 caatgacgca aatgggcgtt ccattgacgt aaatgggcgg taggcgtgcc taatgggagg
     7981 tctatataag caatgctcgt ttagggaac
//
