LOCUS       sequence-448319-        8024 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 8024)
  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 8024)
  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: pEJS1354 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-1 C...
FEATURES             Location/Qualifiers
     source          1..8024
                     /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(1325..1342)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1331..1442
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1693..2072
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2073..2284
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2281..2305
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2445..2577
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2560..2579
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2622..2640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2646..3356
                     /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(3410..3984,3985..3987,3988..3996)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3577..3594)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3804..3823
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4067..4188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4078..4097)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4132..4151
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4323..4341)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4362..4378)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4362..4378)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4375..4397)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4386..4402
                     /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(4410..4416,4417..4434,4435..4440))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4455..4476
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4593..4610)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4764..5352)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4844..4863)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5523..6314,6315..6383))
                     /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     6146..6165
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6384..6488)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6514..6969)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6651..6672)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6863..6882
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7040..8024
                     /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 caagcttgat acatacttct ttacattcca
     1261 atacatactt ctttacattc caatacatac ttctttacat tccaccatag agctcgctga
     1321 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1381 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1441 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1501 ggggaggatt gggaagacaa tagcctaggt agataagtag catggcgggt gatcttcaat
     1561 attggccatt agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc
     1621 cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat
     1681 gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat
     1741 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     1801 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     1861 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     1921 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta
     1981 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt
     2041 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg
     2101 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     2161 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactgcgatc
     2221 gcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2281 agctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt atcacagtta
     2341 aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct gcagtgactc
     2401 tcttaaggta gccttgcaga agttggtcgt gaggcactgg gcaggtaagt atcaaggtta
     2461 caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga agactcttgc
     2521 gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggtg
     2581 tccactccca gttcaattac agctcttaag gctagagtac ttaatacgac tcactatagg
     2641 ctagaatggt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
     2701 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     2761 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     2821 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     2881 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     2941 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     3001 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     3061 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     3121 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca
     3181 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     3241 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     3301 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattgaa
     3361 cgcgtcgagc atgcatctag ggcggccaat tccgcccctc tccccccccc ccctctccct
     3421 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     3481 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     3541 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     3601 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     3661 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     3721 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     3781 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     3841 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     3901 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     3961 tcctttgaaa aacacgatga taagcttgcc acaacccggg atcctctaga gtcgacccgg
     4021 gcggccgctt ccctttagtg agggttaatg cttcgagcag acatgataag atacattgat
     4081 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4141 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4201 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     4261 aacctctaca aatgtggtaa aatccgataa ggatcgatcc gggcggtacc cagcttttgt
     4321 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     4381 tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa
     4441 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4501 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4561 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4621 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4681 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4741 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4801 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4861 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4921 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4981 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5041 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5101 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5161 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5221 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5281 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5341 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5401 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5461 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5521 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5581 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5641 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5701 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5761 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5821 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5881 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5941 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6001 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6061 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6121 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6181 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6241 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6301 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6361 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6421 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6481 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     6541 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6601 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6661 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6721 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6781 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6841 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6901 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6961 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     7021 cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg actcatgcca
     7081 attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg actcgtgcca
     7141 attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg caccatgcca
     7201 attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg gcacggtgcc
     7261 aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc
     7321 aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg
     7381 ccctatgcca atatggctat tggccaggtt caatactatg tattggccct atgccatata
     7441 gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat
     7501 ataccatata tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt
     7561 ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc
     7621 ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca
     7681 ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc
     7741 acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat
     7801 taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat tggcagtact
     7861 cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac
     7921 gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc
     7981 gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaac
//
