LOCUS       sequence-320300-        8555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-bpNLS-dCas13d-bpNLS-ADARdd, ADARdd embeded in loop1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8555)
  AUTHORS   Liu Y, Mao S, Huang S, Li Y, Chen Y, Di M, Huang X, Lv J, Wang X, Ge
            J, Shen S, Zhang X, Liu D, Huang X, Chi T
  TITLE     REPAIRx, a specific yet highly efficient programmable A > I RNA base
            editor.
  JOURNAL   EMBO J. 2020 Nov 16;39(22):e104748. doi: 10.15252/embj.2020104748. 
            Epub 2020 Oct 15.
  PUBMED    33058207
REFERENCE   2  (bases 1 to 8555)
  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: "EMBO J."; 
            date: "2020-11-16"; volume: "39(22):e104748. doi"; pages: " 
            10.15252/embj.2020104748. Epub 2020 Oct 15"
COMMENT     Sequence Label: TC1464
FEATURES             Location/Qualifiers
     source          1..8555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(57..642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(137..156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(813..1604,1605..1673))
                     /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     1436..1455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1674..1778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1853..1872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2044..2423
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2424..2627
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2578..2598
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2672..2691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2672..2690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2768..2788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3386..3406
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4601..4621
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7013..7033
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7040..7105
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7130..7186
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7193..7903
                     /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     7606..7625
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(7942..7959)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7948..8172
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8210..8226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8210..8226)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8223..8245)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8234..8250
                     /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(8258..8264,8265..8282,8283..8288))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8303..8324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8441..8458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
       61 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      121 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      181 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      241 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      301 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      361 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      421 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      481 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      541 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt
      601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1741 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1801 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     1861 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     1921 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     1981 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2041 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2101 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2161 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2221 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2281 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2341 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2401 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2461 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2521 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2581 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2641 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2701 ctggctagcg tttaaactta agcttgccac catgaaacgg actgctgacg gcagcgaatt
     2761 tgagagcccc aagaagaaga ggaaagtcgg atccatcgag aagaagaaga gcttcgccaa
     2821 gggcatggga gtgaagagca ccctggtgtc cggctctaag gtgtacatga ccacatttgc
     2881 tgagggaagc gacgccaggc tggagaagat cgtggagggc gatagcatca gatccgtgaa
     2941 cgagggagag gctttcagcg ccgagatggc tgacaagaac gctggctaca agatcggaaa
     3001 cgccaagttt tcccacccaa agggctacgc cgtggtggct aacaacccac tgtacaccgg
     3061 accagtgcag caggacatgc tgggactgaa ggagacactg gagaagaggt acttcggcga
     3121 gtccgccgac ggaaacgata acatctgcat ccaggtcatc cacaacatcc tggatatcga
     3181 gaagatcctg gctgagtaca tcacaaacgc cgcttacgcc gtgaacaaca tctccggcct
     3241 ggacaaggat atcatcggct tcggaaagtt ttctaccgtg tacacatacg acgagttcaa
     3301 ggatccagag caccaccggg ccgcttttaa caacaacgac aagctgggtt ctaagcgcac
     3361 cgccgatgga tccgagttcg aatcccctaa aaagaaaaga aaggtgggat cccagctgca
     3421 tttaccgcag gttttagctg acgctgtctc acgcctggtc ctgggtaagt ttggtgacct
     3481 gaccgacaac ttctcctccc ctcacgctcg cagaaaagtg ctggctggag tcgtcatgac
     3541 aacaggcaca gatgttaaag atgccaaggt gataagtgtt tctacaggaa caaaatgtat
     3601 taatggtgaa tacatgagtg atcgtggcct tgcattaaat gactgccatg cagaaataat
     3661 atctcggaga tccttgctca gatttcttta tacacaactt gagctttact taaataacaa
     3721 agatgatcaa aaaagatcca tctttcagaa atcagagcga ggggggttta ggctgaagga
     3781 gaatgtccag tttcatctgt acatcagcac ctctccctgt ggagatgcca gaatcttctc
     3841 accacatgag ccaatcctgg aagaaccagc agatagacac ccaaatcgta aagcaagagg
     3901 acagctacgg accaaaatag agtctggtca ggggacgatt ccagtgcgct ccaatgcgag
     3961 catccaaacg tgggacgggg tgctgcaagg ggagcggctg ctcaccatgt cctgcagtga
     4021 caagattgca cgctggaacg tggtgggcat ccagggatca ctgctcagca ttttcgtgga
     4081 gcccatttac ttctcgagca tcatcctggg cagcctttac cacggggacc acctttccag
     4141 ggccatgtac cagcggatct ccaacataga ggacctgcca cctctctaca ccctcaacaa
     4201 gcctttgctc agtggcatca gcaatgcaga agcacggcag ccagggaagg cccccaactt
     4261 cagtgtcaac tggacggtag gcgactccgc tattgaggtc atcaacgcca cgactgggaa
     4321 ggatgagctg ggccgcgcgt cccgcctgtg taagcacgcg ttgtactgtc gctggatgcg
     4381 tgtgcacggc aaggttccct cccacttact acgctccaag attaccaagc ccaacgtgta
     4441 ccatgagtcc aagctggcgg caaaggagta ccaggccgcc aaggcgcgtc tgttcacagc
     4501 cttcatcaag gcggggctgg gggcctgggt ggagaagccc accgagcagg accagttctc
     4561 actcacgaaa agaactgcgg atggatctga gtttgagagt ccaaaaaaga agaggaaggt
     4621 cggttctatc aacgccatca aggctcagta cgacgagttc gataactttc tggataaccc
     4681 caggctgggc tacttcggac aggctttctt ttctaaggag ggcagaaact acatcatcaa
     4741 ctacggaaac gagtgttacg acatcctggc cctgctgagc ggactggccc actgggtggt
     4801 ggccaacaac gaggaggagt ctcggatcag ccgcacctgg ctgtacaacc tggacaagaa
     4861 cctggataac gagtacatct ccacactgaa ctacctgtac gacaggatca ccaacgagct
     4921 gacaaacagc ttctccaaga actctgccgc taacgtgaac tacatcgctg agaccctggg
     4981 catcaaccca gctgagttcg ctgagcagta cttcagattt tccatcatga aggagcagaa
     5041 gaacctgggc ttcaacatca caaagctgag agaagtgatg ctggacagaa aggatatgtc
     5101 cgagatcagg aagaaccaca aggtgttcga ttctatcaga accaaggtgt acacaatgat
     5161 ggactttgtg atctacaggt actacatcga ggaggatgcc aaggtggccg ctgccaacaa
     5221 gagcctgccc gacaacgaga agtctctgag cgagaaggat atcttcgtga tcaacctgag
     5281 aggctccttt aacgacgatc agaaggacgc tctgtactac gatgaggcca acaggatctg
     5341 gagaaagctg gagaacatca tgcacaacat caaggagttc cggggaaaca agacccgcga
     5401 gtacaagaag aaggacgctc caaggctgcc taggatcctg cctgctggaa gggacgtgag
     5461 cgccttcagc aagctgatgt acgccctgac aatgtttctg gacggaaagg agatcaacga
     5521 tctgctgacc acactgatca acaagttcga caacatccag tcttttctga aagtgatgcc
     5581 tctgatcggc gtgaacgcta agttcgtgga ggagtacgcc ttctttaagg acagcgccaa
     5641 gatcgctgat gagctgcggc tgatcaagtc ctttgccagg atgggagagc caatcgctga
     5701 cgctaggaga gctatgtaca tcgatgccat ccggatcctg ggaaccaacc tgtcttacga
     5761 cgagctgaag gctctggccg acaccttcag cctggatgag aacggcaaca agctgaagaa
     5821 gggcaagcac ggaatgcgca acttcatcat caacaacgtg atcagcaaca agcggtttca
     5881 ctacctgatc agatacggcg acccagctca cctgcacgag atcgctaaga acgaggccgt
     5941 ggtgaagttc gtgctgggac ggatcgccga tatccagaag aagcagggcc agaacggaaa
     6001 gaaccagatc gaccgctact acgagacctg catcggcaag gataagggaa agtccgtgtc
     6061 tgagaaggtg gacgctctga ccaagatcat cacaggcatg aactacgacc agttcgataa
     6121 gaagagatct gtgatcgagg acaccggaag ggagaacgcc gagagagaga agtttaagaa
     6181 gatcatcagc ctgtacctga cagtgatcta ccacatcctg aagaacatcg tgaacatcaa
     6241 cgctagatac gtgatcggct tccactgcgt ggagcgcgat gcccagctgt acaaggagaa
     6301 gggatacgac atcaacctga agaagctgga ggagaagggc tttagctccg tgaccaagct
     6361 gtgcgctgga atcgacgaga cagcccccga caagaggaag gatgtggaga aggagatggc
     6421 cgagagagct aaggagagca tcgactccct ggagtctgct aaccctaagc tgtacgccaa
     6481 ctacatcaag tactccgatg agaagaaggc cgaggagttc accaggcaga tcaacagaga
     6541 gaaggccaag accgctctga acgcctacct gaggaacaca aagtggaacg tgatcatccg
     6601 ggaggacctg ctgcgcatcg ataacaagac ctgtacactg ttcgctaaca aggctgtggc
     6661 cctggaggtg gctcgctacg tgcacgccta catcaacgac atcgccgagg tgaactccta
     6721 ctttcagctg taccactaca tcatgcagag gatcatcatg aacgagagat acgagaagtc
     6781 tagcggcaag gtgtctgagt acttcgacgc cgtgaacgat gagaagaagt acaacgatag
     6841 actgctgaag ctgctgtgcg tgcctttcgg atactgtatc ccacggttta agaacctgag
     6901 catcgaggcc ctgttcgacc gcaacgaggc tgccaagttt gataaggaga agaagaaggt
     6961 gagcggcaac tccggttcta agcgcaccgc cgatggatcc gagttcgaat cccctaaaaa
     7021 gaaaagaaag gtgggatccg actacaaaga ccatgacggt gattataaag atcatgatat
     7081 cgattacaag gatgacgatg acaagggatc caaaagaact ggaagcggag ctactaactt
     7141 cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctgtta acatggtgag
     7201 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     7261 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     7321 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     7381 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     7441 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7501 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     7561 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     7621 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     7681 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     7741 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     7801 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     7861 gggccgccac tccaccggcg gcatggacga gctgtacaag taagctcgag cctagagggc
     7921 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     7981 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     8041 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     8101 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     8161 tgggctctat ggtaccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     8221 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     8281 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     8341 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     8401 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     8461 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     8521 ccacagaatc aggggataac gcaggaaaga acatg
//
