LOCUS       sequence-254678-        5212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of AcrIIA4 fused to mCherry with 3x miR-122 
            binding sites in 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5212)
  AUTHORS   Lee J, Mou H, Ibraheim R, Liang SQ, Liu P, Xue W, Sontheimer EJ
  TITLE     Tissue-restricted Genome Editing in vivo Specified by 
            MicroRNA-repressible Anti-CRISPR Proteins.
  JOURNAL   RNA. 2019 Aug 22. pii: rna.071704.119. doi: 10.1261/rna.071704.119.
  PUBMED    31439808
REFERENCE   2  (bases 1 to 5212)
  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. 2019 
            Aug 22. pii: rna.071704.119. doi: 10.1261/rna.071704.119."
COMMENT     Sequence Label: p1149-pCSDest-AcrIIA4-mCherry-miR122BS
FEATURES             Location/Qualifiers
     source          1..5212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..22
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4..21
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             327..350
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             357..377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             390..1100
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(535..553)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     803..822
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(1210..1228)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1233..1367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1257..1276)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1311..1330
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1480..1500)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1480..1498)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1519..1535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1519..1535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1532..1554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1543..1559
                     /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(1567..1573,1574..1591,1592..1597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1612..1633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1750..1767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1921..2509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2001..2020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2680..3471,3472..3540))
                     /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     3303..3322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3541..3645)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3671..4126)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3808..3829)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4020..4039
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4197..5181
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ttgatttagg tgacactata gacggaattg tacccgcggc cgatccaccg gtcgccacca
       61 tgaacatcaa tgatctgatt agagaaataa agaataaaga ctatactgtt aaattgtctg
      121 gaactgacag taatagcata acccaactca tcatcagggt taataatgat ggtaacgaat
      181 atgttataag tgagtccgag aacgaatcta tcgtcgagaa gttcatcagt gccttcaaaa
      241 acggatggaa ccaagagtac gaggatgagg aggaatttta caatgatatg caaacaatca
      301 ctctgaagag cgagcttaac ggcggggatt ataaggatga tgatgataag ggctccccta
      361 agaagaagag aaaggtggga tccggaagta tggtgagcaa gggcgaggag gataacatgg
      421 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
      481 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
      541 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
      601 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
      661 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
      721 ccgtgaccca ggactcctca ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
      781 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
      841 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
      901 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
      961 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     1021 aggactacac catcgtggaa cagtacgaac gcgctgaggg ccgccactcc accggcggca
     1081 tggacgagct gtacaagtga taaagcggcc atcaagcttg acaaacacca ttgtcacact
     1141 ccaacaaaca ccattgtcac actccaacaa acaccattgt cacactccac catagagctc
     1201 gctgatcagc ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     1261 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1321 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1381 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg
     1441 cggcgccaat gcattgggcc cggtacccag cttttgttcc ctttagtgag ggttaattgc
     1501 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1561 tccacacaac atacgagccg ggagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1621 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1681 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1741 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1801 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1861 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1921 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1981 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2041 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2101 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2161 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2221 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2281 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2341 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2401 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2461 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2521 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2581 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2641 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2701 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2761 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2821 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2881 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2941 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3001 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3061 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3121 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3181 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3241 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3301 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3361 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3421 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3481 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3541 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3601 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3661 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3721 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3781 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3841 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3901 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3961 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4021 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4081 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
     4141 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagtcg
     4201 accgccaatt caatatggcg tatatggact catgccaatt caatatggtg gatctggacc
     4261 tgtgccaatt caatatggcg tatatggact cgtgccaatt caatatggtg gatctggacc
     4321 ccagccaatt caatatggcg gacttggcac catgccaatt caatatggcg gacctggcac
     4381 tgtgccaact ggggaggggt ctacttggca cggtgccaag tttgaggagg ggtcttggcc
     4441 ctgtgccaag tccgccatat tgaattggca tggtgccaat aatggcggcc atattggcta
     4501 tatgccagga tcaatatata ggcaatatcc aatatggccc tatgccaata tggctattgg
     4561 ccaggttcaa tactatgtat tggccctatg ccatatagta ttccatatat gggttttcct
     4621 attgacgtag atagcccctc ccaatgggcg gtcccatata ccatatatgg ggcttcctaa
     4681 taccgcccat agccactccc ccattgacgt caatggtctc tatatatggt ctttcctatt
     4741 gacgtcatat gggcggtcct attgacgtat atggcgcctc ccccattgac gtcaattacg
     4801 gtaaatggcc cgcctggctc aatgcccatt gacgtcaata ggaccaccca ccattgacgt
     4861 caatgggatg gctcattgcc cattcatatc cgttctcacg ccccctattg acgtcaatga
     4921 cggtaaatgg cccacttggc agtacatcaa tatctattaa tagtaacttg gcaagtacat
     4981 tactattgga agtacgccag ggtacattgg cagtactccc attgacgtca atggcggtaa
     5041 atggcccgcg atggctgcca agtacatccc cattgacgtc aatggggagg ggcaatgacg
     5101 caaatgggcg ttccattgac gtaaatgggc ggtaggcgtg cctaatggga ggtctatata
     5161 agcaatgctc gtttagggaa ccgccattct gcctggggac gtcggagcaa gc
//
