LOCUS       sequence-448323-        7969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of AcrIIC3-mCherry fusion without microRNA 
            target sites in the 3' UTR as a control 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 7969)
  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 7969)
  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: pEJS1355 CMV-AcrIIC3-FLAGNLS-mCherry CMV-BFP
FEATURES             Location/Qualifiers
     source          1..7969
                     /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(1270..1287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1276..1387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1638..2017
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2018..2229
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2226..2250
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2390..2522
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2505..2524
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2567..2585
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2591..3301
                     /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(3355..3929,3930..3932,3933..3941)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3522..3539)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3749..3768
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4012..4133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4023..4042)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4077..4096
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4268..4286)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4307..4323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4307..4323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4320..4342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4331..4347
                     /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(4355..4361,4362..4379,4380..4385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4400..4421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4538..4555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4709..5297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4789..4808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5468..6259,6260..6328))
                     /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     6091..6110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6329..6433)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6459..6914)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6596..6617)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6808..6827
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6985..7969
                     /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 caagcttatc gataccgtcg actagagctc
     1261 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1321 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1381 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1441 gcaaggggga ggattgggaa gacaatagcc taggtagata agtagcatgg cgggtgatct
     1501 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
     1561 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
     1621 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
     1681 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1741 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1801 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1861 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
     1921 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
     1981 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
     2041 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     2101 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
     2161 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     2221 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
     2281 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
     2341 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
     2401 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
     2461 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     2521 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     2581 ataggctaga atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct
     2641 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     2701 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     2761 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     2821 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     2881 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     2941 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     3001 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     3061 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     3121 cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     3181 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     3241 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     3301 ttgaacgcgt cgagcatgca tctagggcgg ccaattccgc ccctctcccc cccccccctc
     3361 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     3421 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     3481 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     3541 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     3601 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     3661 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     3721 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     3781 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     3841 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     3901 ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccgggatcct ctagagtcga
     3961 cccgggcggc cgcttccctt tagtgagggt taatgcttcg agcagacatg ataagataca
     4021 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4081 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4141 acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca
     4201 agtaaaacct ctacaaatgt ggtaaaatcc gataaggatc gatccgggcg gtacccagct
     4261 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     4321 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccggg agcataaagt
     4381 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4441 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4501 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4561 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4621 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4681 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4741 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4801 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4861 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4921 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4981 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5041 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5101 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5161 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5221 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5281 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5341 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5401 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5461 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5521 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5581 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5641 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5701 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5761 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5821 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5881 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5941 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6001 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6061 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6121 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6181 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6241 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6301 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6361 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6421 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6481 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6541 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6601 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6661 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6721 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6781 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6841 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6901 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     6961 gcgggcctct tcgctattac gccagtcgac cgccaattca atatggcgta tatggactca
     7021 tgccaattca atatggtgga tctggacctg tgccaattca atatggcgta tatggactcg
     7081 tgccaattca atatggtgga tctggacccc agccaattca atatggcgga cttggcacca
     7141 tgccaattca atatggcgga cctggcactg tgccaactgg ggaggggtct acttggcacg
     7201 gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg aattggcatg
     7261 gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg caatatccaa
     7321 tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg gccctatgcc
     7381 atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc aatgggcggt
     7441 cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc attgacgtca
     7501 atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat tgacgtatat
     7561 ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa tgcccattga
     7621 cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca ttcatatccg
     7681 ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag tacatcaata
     7741 tctattaata gtaacttggc aagtacatta ctattggaag tacgccaggg tacattggca
     7801 gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag tacatcccca
     7861 ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt aaatgggcgg
     7921 taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaac
//
