LOCUS       sequence-448316-        8016 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 8016)
  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 8016)
  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: pEJS1351 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-378...
FEATURES             Location/Qualifiers
     source          1..8016
                     /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(3402..3976,3977..3979,3980..3988)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3569..3586)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3796..3815
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4059..4180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4070..4089)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4124..4143
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4315..4333)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4354..4370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4354..4370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4367..4389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4378..4394
                     /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(4402..4408,4409..4426,4427..4432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4447..4468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4585..4602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4756..5344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4836..4855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5515..6306,6307..6375))
                     /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     6138..6157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6376..6480)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6506..6961)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6643..6664)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6855..6874
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7032..8016
                     /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 caagctgcct tctgactcca agtccagtgc
     1261 cttctgactc caagtccagt gccttctgac tccaagtcca gtagctcgct 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 ccccctctcc ctcccccccc
     3421 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3481 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3541 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3601 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     3661 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     3721 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3781 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3841 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3901 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     3961 aaaacacgat gataagcttg ccacaacccg ggatcctcta gagtcgaccc gggcggccgc
     4021 ttccctttag tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg
     4081 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4141 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4201 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta
     4261 caaatgtggt aaaatccgat aaggatcgat ccgggcggta cccagctttt gttcccttta
     4321 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4381 ttatccgctc acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg
     4441 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4501 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4561 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4621 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5161 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5461 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5521 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5581 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5641 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5701 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5761 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5821 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5881 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5941 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6001 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6061 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6121 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6181 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6241 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6301 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6361 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6421 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6481 cacatttccc cgaaaagtgt cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     6541 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     6601 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     6661 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     6721 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     6781 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     6841 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     6901 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6961 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     7021 ctattacgcc agtcgaccgc caattcaata tggcgtatat ggactcatgc caattcaata
     7081 tggtggatct ggacctgtgc caattcaata tggcgtatat ggactcgtgc caattcaata
     7141 tggtggatct ggaccccagc caattcaata tggcggactt ggcaccatgc caattcaata
     7201 tggcggacct ggcactgtgc caactgggga ggggtctact tggcacggtg ccaagtttga
     7261 ggaggggtct tggccctgtg ccaagtccgc catattgaat tggcatggtg ccaataatgg
     7321 cggccatatt ggctatatgc caggatcaat atataggcaa tatccaatat ggccctatgc
     7381 caatatggct attggccagg ttcaatacta tgtattggcc ctatgccata tagtattcca
     7441 tatatgggtt ttcctattga cgtagatagc ccctcccaat gggcggtccc atataccata
     7501 tatggggctt cctaataccg cccatagcca ctcccccatt gacgtcaatg gtctctatat
     7561 atggtctttc ctattgacgt catatgggcg gtcctattga cgtatatggc gcctccccca
     7621 ttgacgtcaa ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt caataggacc
     7681 acccaccatt gacgtcaatg ggatggctca ttgcccattc atatccgttc tcacgccccc
     7741 tattgacgtc aatgacggta aatggcccac ttggcagtac atcaatatct attaatagta
     7801 acttggcaag tacattacta ttggaagtac gccagggtac attggcagta ctcccattga
     7861 cgtcaatggc ggtaaatggc ccgcgatggc tgccaagtac atccccattg acgtcaatgg
     7921 ggaggggcaa tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa
     7981 tgggaggtct atataagcaa tgctcgttta gggaac
//
