LOCUS       sequence-443101-        6999 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of CMV-TadA8e-NLS-dCas13d-NLS in mammalian cells (DECOR 
            expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6999)
  AUTHORS   Yan H, Tang W
  TITLE     Programmed RNA editing with an evolved bacterial adenosine 
            deaminase.
  JOURNAL   Nat Chem Biol. 2024 Jul 5. doi: 10.1038/s41589-024-01661-x.
  PUBMED    38969862
REFERENCE   2  (bases 1 to 6999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2024 Jul 5. doi: 10.1038/s41589-024-01661-x."
COMMENT     Sequence Label: DECOR
FEATURES             Location/Qualifiers
     source          1..6999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     574..593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(812..916)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(995..1014)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1186..1565
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1566..1769
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1720..1740
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1811..1830
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1811..1829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2443..2463
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2473..5370
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             5380..5400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5416..5439
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5448..5465
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5488..5505)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5494..5718
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5789..5805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5789..5805)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5802..5824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5813..5829
                     /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(5837..5843,5844..5861,5862..5867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5882..5903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6020..6037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6191..6779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6271..6290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6950..6999,1..742,743..811))
                     /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"
ORIGIN
        1 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
       61 catctggccc cagtgctgca atgataccgc ggctaccacg ctcaccggct ccagatttat
      121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
      181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
      241 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      541 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      901 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
      961 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
     1021 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     1081 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     1141 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     1201 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     1261 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     1321 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     1381 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1441 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     1501 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1561 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1621 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1681 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1741 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagagat
     1801 ccgcggccgc taatacgact cactataggg agagccgcca ccatgtctga ggtggagttt
     1861 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg cgatgagagg
     1921 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
     1981 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
     2041 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
     2101 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
     2161 aggaactcaa aacgcggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
     2221 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
     2281 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
     2341 aactctggag gatctagcgg aggatcctct ggcagcgaga caccaggaac aagcgagtca
     2401 gcaacaccag agagcagtgg cggcagcagc ggcggcagca gccccaagaa gaagagaaag
     2461 gtggaggcca gcatcgaaaa aaaaaagtcc ttcgccaagg gcatgggcgt gaagtccaca
     2521 ctcgtgtccg gctccaaagt gtacatgaca accttcgccg aaggcagcga cgccaggctg
     2581 gaaaagatcg tggagggcga cagcatcagg agcgtgaatg agggcgaggc cttcagcgct
     2641 gaaatggccg ataaaaacgc cggctataag atcggcaacg ccaaattcag ccatcctaag
     2701 ggctacgccg tggtggctaa caaccctctg tatacaggac ccgtccagca ggatatgctc
     2761 ggcctgaagg aaactctgga aaagaggtac ttcggcgaga gcgctgatgg caatgacaat
     2821 atttgtatcc aggtgatcca taacatcctg gacattgaaa aaatcctcgc cgaatacatt
     2881 accaacgccg cctacgccgt caacaatatc tccggcctgg ataaggacat tattggattc
     2941 ggcaagttct ccacagtgta tacctacgac gaattcaaag accccgagca ccatagggcc
     3001 gctttcaaca ataacgataa gctcatcaac gccatcaagg cccagtatga cgagttcgac
     3061 aacttcctcg ataaccccag actcggctat ttcggccagg cctttttcag caaggagggc
     3121 agaaattaca tcatcaatta cggcaacgaa tgctatgaca ttctggccct cctgagcgga
     3181 ctggcgcact gggtggtcgc taacaacgaa gaagagtcca ggatctccag gacctggctc
     3241 tacaacctcg ataagaacct cgacaacgaa tacatctcca ccctcaacta cctctacgac
     3301 aggatcacca atgagctgac caactccttc tccaagaact ccgccgccaa cgtgaactat
     3361 attgccgaaa ctctgggaat caaccctgcc gaattcgccg aacaatattt cagattcagc
     3421 attatgaaag agcagaaaaa cctcggattc aatatcacca agctcaggga agtgatgctg
     3481 gacaggaagg atatgtccga gatcaggaaa aatcataagg tgttcgactc catcaggacc
     3541 aaggtctaca ccatgatgga ctttgtgatt tataggtatt acatcgaaga ggatgccaag
     3601 gtggctgccg ccaataagtc cctccccgat aatgagaagt ccctgagcga gaaggatatc
     3661 tttgtgatta acctgagggg ctccttcaac gacgaccaga aggatgccct ctactacgat
     3721 gaagctaata gaatttggag aaagctcgaa aatatcatgc acaacatcaa ggaatttagg
     3781 ggaaacaaga caagagagta taagaagaag gacgccccta gactgcccag aatcctgccc
     3841 gctggccgtg atgtttccgc cttcagcaaa ctcatgtatg ccctgaccat gttcctggat
     3901 ggcaaggaga tcaacgacct cctgaccacc ctgattaata aattcgataa catccagagc
     3961 ttcctgaagg tgatgcctct catcggagta aacgctaagt tcgtggagga atacgccttt
     4021 ttcaaagact ccgccaagat cgccgatgag ctgaggctga tcaagtcctt cgctagaatg
     4081 ggagaaccta ttgccgatgc caggagggcc atgtatatcg acgccatccg tattttagga
     4141 accaacctgt cctatgatga gctcaaggcc ctcgccgaca ccttttccct ggacgagaac
     4201 ggaaacaagc tcaagaaagg caagcacggc atgagaaatt tcattattaa taacgtgatc
     4261 agcaataaaa ggttccacta cctgatcaga tacggtgatc ctgcccacct ccatgagatc
     4321 gccaaaaacg aggccgtggt gaagttcgtg ctcggcagga tcgctgacat ccagaaaaaa
     4381 cagggccaga acggcaagaa ccagatcgac aggtactacg aaacttgtat cggaaaggat
     4441 aagggcaaga gcgtgagcga aaaggtggac gctctcacaa agatcatcac cggaatgaac
     4501 tacgaccaat tcgacaagaa aaggagcgtc attgaggaca ccggcaggga aaacgccgag
     4561 agggagaagt ttaaaaagat catcagcctg tacctcaccg tgatctacca catcctcaag
     4621 aatattgtca atatcaacgc caggtacgtc atcggattcc attgcgtcga gcgtgatgct
     4681 caactgtaca aggagaaagg ctacgacatc aatctcaaga aactggaaga gaagggattc
     4741 agctccgtca ccaagctctg cgctggcatt gatgaaactg cccccgataa gagaaaggac
     4801 gtggaaaagg agatggctga aagagccaag gagagcattg acagcctgga gagcgccaac
     4861 cccaagctgt atgccaatta catcaaatac agcgacgaga agaaagccga ggagttcacc
     4921 aggcagatta acagggagaa ggccaaaacc gccctgaacg cctacctgag gaacaccaag
     4981 tggaatgtga tcatcaggga ggacctcctg agaattgaca acaagacatg taccctgttc
     5041 gccaacaagg ccgtcgccct ggaagtggcc aggtatgtcc acgcctatat caacgacatt
     5101 gccgaggtca attcctactt ccaactgtac cattacatca tgcagagaat tatcatgaat
     5161 gagaggtacg agaaaagcag cggaaaggtg tccgagtact tcgacgctgt gaatgacgag
     5221 aagaagtaca acgataggct cctgaaactg ctgtgtgtgc ctttcggcta ctgtatcccc
     5281 aggtttaaga acctgagcat cgaggccctg ttcgatagga acgaggccgc caagttcgac
     5341 aaggagaaaa agaaggtgtc cggcaattcc ggatccggac ctaagaaaaa gaggaaggtg
     5401 ggatccgcgg ccgctgatta caaggacgac gatgacaagt aaccggtcat catcaccatc
     5461 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5521 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5581 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5641 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     5701 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga
     5761 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5821 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct
     5881 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5941 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6001 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6061 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6121 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6181 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6241 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6301 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6361 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6421 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6481 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6541 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6601 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6661 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6721 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6781 aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag
     6841 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6901 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6961 taatcagtga ggcacctatc tcagcgatct gtctatttc
//
