LOCUS       sequence-448313-        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: pEJS1350 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-92a...
FEATURES             Location/Qualifiers
     source          1..8015
                     /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(1317..1334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1323..1434
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1685..2064
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2065..2276
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2273..2297
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2437..2569
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2552..2571
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2614..2632
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2638..3348
                     /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(3401..3975,3976..3978,3979..3987)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3568..3585)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3795..3814
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4058..4179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4069..4088)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4123..4142
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4314..4332)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4353..4369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4353..4369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4366..4388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4377..4393
                     /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(4401..4407,4408..4425,4426..4431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4446..4467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4584..4601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4755..5343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4835..4854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5514..6305,6306..6374))
                     /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     6137..6156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6375..6479)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6505..6960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6642..6663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6854..6873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7031..8015
                     /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 caagctacag gccgggacaa gtgcaataac
     1261 aggccgggac aagtgcaata acaggccggg acaagtgcaa taagctcgct gatcagcctc
     1321 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1381 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1441 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1501 ttgggaagac aatagcctag gtagataagt agcatggcgg gtgatcttca atattggcca
     1561 ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat
     1621 acgttgtatc tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca
     1681 tgttggcatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     1741 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1801 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1861 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1921 catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc
     1981 gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca gtacatctac
     2041 gtattagtca tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga
     2101 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     2161 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactgcga tcgcccgccc
     2221 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
     2281 tagtgaaccg tcagatcact agaagcttta ttgcggtagt ttatcacagt taaattgcta
     2341 acgcagtcag tgcttctgac acaacagtct cgaacttaag ctgcagtgac tctcttaagg
     2401 tagccttgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag
     2461 gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga
     2521 taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc
     2581 cagttcaatt acagctctta aggctagagt acttaatacg actcactata ggctagaatg
     2641 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
     2701 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     2761 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     2821 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     2881 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     2941 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     3001 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     3061 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     3121 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca
     3181 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     3241 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     3301 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaattg aacgcgtcga
     3361 gcatgcatct agggcggcca attccgcccc tctccccccc cccctctccc tccccccccc
     3421 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     3481 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     3541 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     3601 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     3661 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     3721 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     3781 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     3841 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     3901 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     3961 aaacacgatg ataagcttgc cacaacccgg gatcctctag agtcgacccg ggcggccgct
     4021 tccctttagt gagggttaat gcttcgagca gacatgataa gatacattga tgagtttgga
     4081 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4141 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4201 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     4261 aaatgtggta aaatccgata aggatcgatc cgggcggtac ccagcttttg ttccctttag
     4321 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4381 tatccgctca caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt
     4441 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4501 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4561 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4621 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4681 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4741 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4801 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4861 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4921 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4981 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5041 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5101 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5161 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5221 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5281 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5341 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5401 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5461 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5521 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5581 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5641 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5701 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5761 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5821 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5881 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5941 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6001 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6061 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6121 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6181 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6241 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6301 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6361 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6421 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6481 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6541 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6721 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6781 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6841 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6901 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6961 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7021 tattacgcca gtcgaccgcc aattcaatat ggcgtatatg gactcatgcc aattcaatat
     7081 ggtggatctg gacctgtgcc aattcaatat ggcgtatatg gactcgtgcc aattcaatat
     7141 ggtggatctg gaccccagcc aattcaatat ggcggacttg gcaccatgcc aattcaatat
     7201 ggcggacctg gcactgtgcc aactggggag gggtctactt ggcacggtac caagtttgag
     7261 gaggggtctt ggccctgtgc caagtccgcc atattgaatt ggcatggtgc caataatggc
     7321 ggccatattg gctatatgcc aggatcaata tataggcaat atccaatatg gccctatgcc
     7381 aatatggcta ttggccaggt tcaatactat gtattggccc tatgccatat agtattccat
     7441 atatgggttt tcctattgac gtagatagcc cctcccaatg ggcggtccca tataccatat
     7501 atggggcttc ctaataccgc ccatagccac tcccccattg acgtcaatgg tctctatata
     7561 tggtctttcc tattgacgtc atatgggcgg tcctattgac gtatatggcg cctcccccat
     7621 tgacgtcaat tacggtaaat ggcccgcctg gctcaatgcc cattgacgtc aataggacca
     7681 cccaccattg acgtcaatgg gatggctcat tgcccattca tatccgttct cacgccccct
     7741 attgacgtca atgacggtaa atggcccact tggcagtaca tcaatatcta ttaatagtaa
     7801 cttggcaagt acattactat tggaagtacg ccagggtaca ttggcagtac tcccattgac
     7861 gtcaatggcg gtaaatggcc cgcgatggct gccaagtaca tccccattga cgtcaatggg
     7921 gaggggcaat gacgcaaatg ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat
     7981 gggaggtcta tataagcaat gctcgtttag ggaac
//
