LOCUS       sequence-299887-        4860 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  TetON-APEX2-V5-BPNLS-dRfxCas13d-dsRBD-BPNLS-P2A-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4860)
  AUTHORS   Han S, Zhao BS, Myers SA, Carr SA, He C, Ting AY
  TITLE     RNA-protein interaction mapping via MS2- or Cas13-based APEX 
            targeting.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Aug 24. pii: 2006617117. doi: 
            10.1073/pnas.2006617117.
  PUBMED    32839320
REFERENCE   2  (bases 1 to 4860)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Aug 24. pii: 2006617117. doi: 
            10.1073/pnas.2006617117."
COMMENT     Sequence Label: dCas13d-dsRBD-APEX2
FEATURES             Location/Qualifiers
     source          1..4860
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..750
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     CDS             751..792
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             832..852
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             862..3759
                     /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             4048..4068
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4078..4134
                     /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             join(4141..4143,4144..4146,4147..4860)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4141..4143,4144..4146,4147..4857)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4186..4207)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4447..4466)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4794..4815
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atgggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
       61 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
      121 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
      181 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
      241 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
      301 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
      361 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat
      421 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
      481 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
      541 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
      601 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
      661 gttgacaaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
      721 aagctttccg agcttgggtt tgctgatgcc ggcaagccca tccccaaccc cctgctgggc
      781 ctggacagca ccggtagcaa gcggactgct gatggcagtg aatttgagag ccccaagaag
      841 aagagaaagg tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg catgggcgtg
      901 aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga aggcagcgac
      961 gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga gggcgaggcc
     1021 ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc caaattcagc
     1081 catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc cgtccagcag
     1141 gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag cgctgatggc
     1201 aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa aatcctcgcc
     1261 gaatacatta ccaacgccgc ctacgccgtc aacaatatct ccggcctgga taaggacatt
     1321 attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga ccccgagcac
     1381 catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc ccagtatgac
     1441 gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc ctttttcagc
     1501 aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat tctggccctc
     1561 ctgagcggac tggcgcactg ggtggtcgct aacaacgaag aagagtccag gatctccagg
     1621 acctggctct acaacctcga taagaacctc gacaacgaat acatctccac cctcaactac
     1681 ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc cgccgccaac
     1741 gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga acaatatttc
     1801 agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa gctcagggaa
     1861 gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt gttcgactcc
     1921 atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta catcgaagag
     1981 gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc cctgagcgag
     2041 aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa ggatgccctc
     2101 tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca caacatcaag
     2161 gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag actgcccaga
     2221 atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc cctgaccatg
     2281 ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa attcgataac
     2341 atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt cgtggaggaa
     2401 tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat caagtccttc
     2461 gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga cgccatccgt
     2521 attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac cttttccctg
     2581 gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt cattattaat
     2641 aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc tgcccacctc
     2701 catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat cgctgacatc
     2761 cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga aacttgtatc
     2821 ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa gatcatcacc
     2881 ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac cggcagggaa
     2941 aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt gatctaccac
     3001 atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca ttgcgtcgag
     3061 cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa actggaagag
     3121 aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc ccccgataag
     3181 agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga cagcctcgag
     3241 agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa gaaagccgag
     3301 gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc ctacctgagg
     3361 aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa caagacatgt
     3421 accctgttcg caaacaaggc cgtcgccctg gaagtggcca ggtatgtcca cgcctatatc
     3481 aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat gcagagaatt
     3541 atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt cgacgctgtg
     3601 aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc tttcggctac
     3661 tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa cgaggccgcc
     3721 aagttcgaca aggagaaaaa gaaggtgtcc ggcaattccg gatccggagc aggcgatttg
     3781 tccgcaggtt tcttcatgga agaactgaac acttatcgcc agaaacaggg agttgtgctg
     3841 aagtaccaag agttgccgaa ctcaggccca cctcacgacc gaagattcac ttttcaagta
     3901 attatcgatg gtagagagtt cccagaaggc gaaggccgaa gtaagaaaga agcgaagaac
     3961 gcagccgcta aattggcagt cgaaatcctc aataaagaaa agaaagcggg atccaagcgc
     4021 accgccgatg gttccgagtt cgaaagcccc aagaaaaagc gcaaggtcgg aagcggagct
     4081 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctgaattc
     4141 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4201 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4261 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     4321 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4381 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4441 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4501 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4561 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4621 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4681 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4741 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4801 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
//
