LOCUS       sequence-448321-        8015 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 8015)
  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 8015)
  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: pEJS1386 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-107...
FEATURES             Location/Qualifiers
     source          1..8015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..825
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     822..846
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          986..1118
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1101..1120
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1163..1181
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1187..1897
                     /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(1951..2525,2526..2528,2529..2537)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2118..2135)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2345..2364
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2608..2729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2619..2638)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2673..2692
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2864..2882)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2903..2919)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2903..2919)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2916..2938)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2927..2943
                     /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(2951..2957,2958..2975,2976..2981))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2996..3017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3134..3151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3305..3893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3385..3404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4064..4855,4856..4924))
                     /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     4687..4706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4925..5029)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5055..5510)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5192..5213)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5404..5423
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5581..6565
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        6600..6618
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6600..6617
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             7010..7033
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7040..7060
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7073..7783
                     /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(7218..7236)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7486..7505
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(7881..7898)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7887..7998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
       61 atagcctagg tagataagta gcatggcggg tgatcttcaa tattggccat tagccatatt
      121 attcattggt tatatagcat aaatcaatat tggctattgg ccattgcata cgttgtatct
      181 atatcataat atgtacattt atattggctc atgtccaata tgaccgccat gttggcattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactgcgat cgcccgcccc gttgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
      841 cagatcacta gaagctttat tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt
      901 gcttctgaca caacagtctc gaacttaagc tgcagtgact ctcttaaggt agccttgcag
      961 aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt acaagacagg tttaaggaga
     1021 ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat
     1081 tggtcttact gacatccact ttgcctttct ctccacaggt gtccactccc agttcaatta
     1141 cagctcttaa ggctagagta cttaatacga ctcactatag gctagaatgg tgtctaaggg
     1201 cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa
     1261 ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
     1321 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag
     1381 cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa
     1441 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
     1501 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
     1561 cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
     1621 ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc
     1681 cctgaagctc gtgggcggga gccatctgat cgcaaacgcc aagaccacat atagatccaa
     1741 gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag
     1801 aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata
     1861 ctgcgacctc cctagcaaac tggggcacaa gcttaattga acgcgtcgag catgcatcta
     1921 gggcggccaa ttccgcccct ctcccccccc cccctctccc tccccccccc ctaacgttac
     1981 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     2041 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2101 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     2161 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     2221 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     2281 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     2341 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2401 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     2461 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     2521 ataagcttgc cacaacccgg gatcctctag agtcgacccg ggcggccgct tccctttagt
     2581 gagggttaat gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     2641 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2701 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     2761 aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     2821 aaatccgata aggatcgatc cgggcggtac ccagcttttg ttccctttag tgagggttaa
     2881 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2941 caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag
     3001 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3061 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3121 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3181 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3241 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3301 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3361 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3421 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3481 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3541 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3601 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3661 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3721 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3781 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3841 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3901 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3961 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4021 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4081 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4141 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4201 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4261 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4321 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4381 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4441 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4501 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4561 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4621 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4681 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4741 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4801 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4861 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4921 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4981 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5041 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5101 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5161 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5221 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5281 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5341 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5401 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5461 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     5521 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     5581 gtcgaccgcc aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg
     5641 gacctgtgcc aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg
     5701 gaccccagcc aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacctg
     5761 gcactgtgcc aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt
     5821 ggccctgtgc caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg
     5881 gctatatgcc aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta
     5941 ttggccaggt tcaatactat gtattggccc tatgccatat agtattccat atatgggttt
     6001 tcctattgac gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc
     6061 ctaataccgc ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc
     6121 tattgacgtc atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat
     6181 tacggtaaat ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg
     6241 acgtcaatgg gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca
     6301 atgacggtaa atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt
     6361 acattactat tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg
     6421 gtaaatggcc cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat
     6481 gacgcaaatg ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta
     6541 tataagcaat gctcgtttag ggaaccgcca ttctgcctgg ggacgtcgga gcaagcttga
     6601 tttaggtgac actatagacg gaattgtacc cgcggccgat ccaccggtcg ccaccatgtt
     6661 caaacgggcc attattttca ccagcttcaa cggcttcgag aaggtcagtc gcacggaaaa
     6721 acggcgcctt gccaagataa ttaacgccag agttagtata atcgacgagt atcttcgcgc
     6781 caaagacacc aacgcctcct tggacggaca gtatagagca ttccttttca atgatgagtc
     6841 accagcgatg accgaattcc tcgctaagct caaggcgttc gcagaaagct gtactggtat
     6901 aagcattgat gcgtgggaga ttgaagagag cgagtatgtt cgattgcccg tcgaacggag
     6961 agatttcctt gcggcggcca atggcaaaga aattttcaaa attggcgggg attataagga
     7021 tgatgatgat aagggctccc ctaagaagaa gagaaaggtg ggatccggaa gtatggtgag
     7081 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     7141 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     7201 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     7261 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     7321 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7381 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tcactgcagg acggcgagtt
     7441 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     7501 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     7561 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactactacg ctgaggtcaa
     7621 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     7681 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgctga
     7741 gggccgccac tccaccggcg gcatggacga gctgtacaag tgataaagcg gccatcaagc
     7801 ttatagccct gtacaatgct gctatagccc tgtacaatgc tgctatagcc ctgtacaatg
     7861 ctgctgagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     7921 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     7981 aaatgaggaa attgcatcgc attgtctgag taggt
//
