LOCUS       sequence-477189-        8019 bp    DNA     circular SYN 08-AUG-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 8019)
  TITLE     Absolute Quantification of Mammalian MicroRNAs for Therapeutic RNA 
            Cleavage and Detargeting
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8019)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pEJS2787 CMV-AcrIIC3-FLAGNLS-mCherry-3xmiR-221...
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1165..1286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1202..1221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1256..1275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    join(1357..1365,1366..1368,1369..1943)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1530..1549)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1759..1776
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(1997..2707)
                     /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"
     promoter        complement(2713..2731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2774..2793)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2776..2908
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(3048..3072)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(3069..3280)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        3281..3660
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    3911..4022
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4011..4028
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4130..4840)
                     /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(4408..4427)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4677..4695
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4853..4873)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4880..4903)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(5295..5313)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5296..5313)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(5348..6332)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      6403..6858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6490..6509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6700..6721
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6884..6988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6989..7057,7058..7849)
                     /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     complement(7207..7226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgctccc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggtaccgc ccggatcgat ccttatcgga ttttaccaca
     1081 tttgtagagg ttttacttgc tttaaaaaac ctcccacatc tccccctgaa cctgaaacat
     1141 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     1201 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1261 ttgtccaaac tcatcaatgt atcttatcat gtctgctcga agcattaacc ctcactaaag
     1321 ggaagcggcc gcccgggtcg actctagagg atcccgggtt gtggcaagct tatcatcgtg
     1381 tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt tttaacctcg
     1441 actaaacaca tgtaaagcat gtgcaccgag gccccagatc agatcccata caatggggta
     1501 ccttctgggc atccttcagc cccttgttga atacgcttga ggagagccat ttgactcttt
     1561 ccacaactat ccaactcaca acgtggcact ggggttgtgc cgcctttgca ggtgtatctt
     1621 atacacgtgg cttttggccg cagaggcacc tgtcgccagg tggggggttc cgctgcctgc
     1681 aaagggtcgc tacagacgtt gtttgtcttc aagaagcttc cagaggaact gcttccttca
     1741 cgacattcaa cagaccttgc attcctttgg cgagagggga aagaccccta ggaatgctcg
     1801 tcaagaagac agggccaggt ttccgggccc tcacattgcc aaaagacggc aatatggtgg
     1861 aaaataacat atagacaaac gcacaccggc cttattccaa gcggcttcgg ccagtaacgt
     1921 tagggggggg ggagggagag gggggggggg gagaggggcg gaattggccg ccctagatgc
     1981 atgctcgacg cgttcaatta agcttgtgcc ccagtttgct agggaggtcg cagtatctgg
     2041 ccactgccac ctcgtgctgc tcgacgtagg tctcgttgtt ggcctccttg attctttcca
     2101 gtctgtagtc cacatagtag acgccaggca tcttgaggtt cttagcgggt ttcttggatc
     2161 tatatgtggt cttggcgttt gcgatcagat ggctcccgcc cacgagcttc agggccatgt
     2221 cgtttctgcc ttccaggccg ccgtcagcgg ggtacagcgt ctcggtgaag gcctcccagc
     2281 cgagtgtttt cttctgcatc acagggccgt tggatgtgaa gttcacccct ctgatcttga
     2341 cgttgtagat gaggcagccg tcctggaggc tggtgtcctg ggtagcggtc agcacgcccc
     2401 cgtcttcgta tgtggtgact ctctcccatg tgaagccctc agggaaggac tgcttgaaga
     2461 agtcggggat gccctgggtg tggttgatga aggtcttgct gccgtagagg aagctagtag
     2521 ccaggatgtc gaaggcgaag gggagagggc cgccctcgac caccttgatt ctcatggtct
     2581 gggtgccctc gtagggcttg ccttcgccct cggatgtgca cttgaagtga tggttgtcca
     2641 cggtgccctc catgtacagc ttcatgtgca tgttctcctt aatcagctct tcgcccttag
     2701 acaccattct agcctatagt gagtcgtatt aagtactcta gccttaagag ctgtaattga
     2761 actgggagtg gacacctgtg gagagaaagg caaagtggat gtcagtaaga ccaataggtg
     2821 cctatcagaa acgcaagagt cttctctgtc tcgacaagcc cagtttctat tggtctcctt
     2881 aaacctgtct tgtaaccttg atacttacct gcccagtgcc tcacgaccaa cttctgcaag
     2941 gctaccttaa gagagtcact gcagcttaag ttcgagactg ttgtgtcaga agcactgact
     3001 gcgttagcaa tttaactgtg ataaactacc gcaataaagc ttctagtgat ctgacggttc
     3061 actaaacgag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     3121 aacggggcgg gcgatcgcag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     3181 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     3241 gctatccacg cccattggtg tactgccaaa accgcatcac catggtaata gcgatgacta
     3301 atacgtagat gtactgccaa gtaggaaagt cccgtaaggt catgtactgg gcataatgcc
     3361 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgg acttggcata tgatacactt
     3421 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     3481 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     3541 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     3601 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgcc
     3661 aacatggcgg tcatattgga catgagccaa tataaatgta catattatga tatagataca
     3721 acgtatgcaa tggccaatag ccaatattga tttatgctat ataaccaatg aataatatgg
     3781 ctaatggcca atattgaaga tcacccgcca tgctacttat ctacctaggc tattgtcttc
     3841 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     3901 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     3961 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     4021 agtcgaggct gatcagcgag ctgctagagc tacattgtct gctgggttag ctacattgtc
     4081 tgctgggtta gctacattgt ctgctgggtt tcgagtgatg gccgctttat cacttgtaca
     4141 gctcgtccat gccgccggtg gagtggcggc cctcagcgcg ttcgtactgt tccacgatgg
     4201 tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag gcgccgggca
     4261 gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtagtag tggccgccgt
     4321 ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc
     4381 gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga
     4441 agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagt gaggagtcct
     4501 gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct
     4561 cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg
     4621 agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccacccttgg
     4681 tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga
     4741 tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct
     4801 tgatgatggc catgttatcc tcctcgccct tgctcaccat acttccggat cccacctttc
     4861 tcttcttctt aggggagccc ttatcatcat catccttata atccccgcca attttgaaaa
     4921 tttctttgcc attggccgcc gcaaggaaat ctctccgttc gacgggcaat cgaacatact
     4981 cgctctcttc aatctcccac gcatcaatgc ttataccagt acagctttct gcgaacgcct
     5041 tgagcttagc gaggaattcg gtcatcgctg gtgactcatc attgaaaagg aatgctctat
     5101 actgtccgtc caaggaggcg ttggtgtctt tggcgcgaag atactcgtcg attatactaa
     5161 ctctggcgtt aattatcttg gcaaggcgcc gtttttccgt gcgactgacc ttctcgaagc
     5221 cgttgaagct ggtgaaaata atggcccgtt tgaacatggt ggcgaccggt ggatcggccg
     5281 cgggtacaat tccgtctata gtgtcaccta aatcaagctt gctccgacgt ccccaggcag
     5341 aatggcggtt ccctaaacga gcattgctta tatagacctc ccattaggca cgcctaccgc
     5401 ccatttacgt caatggaacg cccatttgcg tcattgcccc tccccattga cgtcaatggg
     5461 gatgtacttg gcagccatcg cgggccattt accgccattg acgtcaatgg gagtactgcc
     5521 aatgtaccct ggcgtacttc caatagtaat gtacttgcca agttactatt aatagatatt
     5581 gatgtactgc caagtgggcc atttaccgtc attgacgtca atagggggcg tgagaacgga
     5641 tatgaatggg caatgagcca tcccattgac gtcaatggtg ggtggtccta ttgacgtcaa
     5701 tgggcattga gccaggcggg ccatttaccg taattgacgt caatggggga ggcgccatat
     5761 acgtcaatag gaccgcccat atgacgtcaa taggaaagac catatataga gaccattgac
     5821 gtcaatgggg gagtggctat gggcggtatt aggaagcccc atatatggta tatgggaccg
     5881 cccattggga ggggctatct acgtcaatag gaaaacccat atatggaata ctatatggca
     5941 tagggccaat acatagtatt gaacctggcc aatagccata ttggcatagg gccatattgg
     6001 atattgccta tatattgatc ctggcatata gccaatatgg ccgccattat tggcaccatg
     6061 ccaattcaat atggcggact tggcacaggg ccaagacccc tcctcaaact tggcaccgtg
     6121 ccaagtagac ccctccccag ttggcacagt gccaggtccg ccatattgaa ttggcatggt
     6181 gccaagtccg ccatattgaa ttggctgggg tccagatcca ccatattgaa ttggcacgag
     6241 tccatatacg ccatattgaa ttggcacagg tccagatcca ccatattgaa ttggcatgag
     6301 tccatatacg ccatattgaa ttggcggtcg actggcgtaa tagcgaagag gcccgcaccg
     6361 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     6421 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     6481 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6541 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6601 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6661 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6721 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     6781 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6841 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6901 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6961 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     7021 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     7081 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     7141 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     7201 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     7261 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     7321 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     7381 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     7441 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     7501 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     7561 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     7621 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     7681 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     7741 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7801 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7861 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7921 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7981 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
