LOCUS       sequence-298655-        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid to carry out IVT of RfxCas13d (human codon-optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  AUTHORS   Kushawah G, Hernandez-Huertas L, Abugattas-Nunez Del Prado J, 
            Martinez-Morales JR, DeVore ML, Hassan H, Moreno-Sanchez I, 
            Tomas-Gallardo L, Diaz-Moscoso A, Monges DE, Guelfo JR, Theune WC, 
            Brannan EO, Wang W, Corbin TJ, Moran AM, Sanchez Alvarado A, 
            Malaga-Trillo E, Takacs CM, Bazzini AA, Moreno-Mateos MA
  TITLE     CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.
  JOURNAL   Dev Cell. 2020 Sep 28;54(6):805-817.e7. doi: 
            10.1016/j.devcel.2020.07.013. Epub 2020 Aug 7.
  PUBMED    32768421
REFERENCE   2  (bases 1 to 6116)
  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"; doi: 
            "10.1016/j.devcel.2020.07.013"; journalName: "Dev Cell"; date: 
            "2020-09-28- 28"; volume: "54"; issue: "6"; pages: "805-817.e7"
COMMENT     Sequence Label: pT3TS-RfxCas13d-HA
FEATURES             Location/Qualifiers
     source          1..6116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      102..111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             126..3023
                     /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
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3042..3068
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     3'UTR           3093..3218
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(3304..3323)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3305..3323)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3330..3347)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3330..3346)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3339..3361)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3488..3943)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3625..3646)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3837..3856
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3970..4074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4075..4143,4144..4935)
                     /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(4293..4312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5106..5694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5595..5614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5848..5865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5982..6003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6018..6023,6024..6041,6042..6048)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6056..6072
                     /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     6061..6083
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6080..6096
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6080..6096
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg gcgagcgagg
      121 ccagcatcga aaaaaaaaag tccttcgcca agggcatggg cgtgaagtcc acactcgtgt
      181 ccggctccaa agtgtacatg acaaccttcg ccgaaggcag cgacgccagg ctggaaaaga
      241 tcgtggaggg cgacagcatc aggagcgtga atgagggcga ggccttcagc gctgaaatgg
      301 ccgataaaaa cgccggctat aagatcggca acgccaaatt cagccatcct aagggctacg
      361 ccgtggtggc taacaaccct ctgtatacag gacccgtcca gcaggatatg ctcggcctga
      421 aggaaactct ggaaaagagg tacttcggcg agagcgctga tggcaatgac aatatttgta
      481 tccaggtgat ccataacatc ctggacattg aaaaaatcct cgccgaatac attaccaacg
      541 ccgcctacgc cgtcaacaat atctccggcc tggataagga cattattgga ttcggcaagt
      601 tctccacagt gtatacctac gacgaattca aagaccccga gcaccatagg gccgctttca
      661 acaataacga taagctcatc aacgccatca aggcccagta tgacgagttc gacaacttcc
      721 tcgataaccc cagactcggc tatttcggcc aggccttttt cagcaaggag ggcagaaatt
      781 acatcatcaa ttacggcaac gaatgctatg acattctggc cctcctgagc ggactgaggc
      841 actgggtggt ccataacaac gaagaagagt ccaggatctc caggacctgg ctctacaacc
      901 tcgataagaa cctcgacaac gaatacatct ccaccctcaa ctacctctac gacaggatca
      961 ccaatgagct gaccaactcc ttctccaaga actccgccgc caacgtgaac tatattgccg
     1021 aaactctggg aatcaaccct gccgaattcg ccgaacaata tttcagattc agcattatga
     1081 aagagcagaa aaacctcgga ttcaatatca ccaagctcag ggaagtgatg ctggacagga
     1141 aggatatgtc cgagatcagg aaaaatcata aggtgttcga ctccatcagg accaaggtct
     1201 acaccatgat ggactttgtg atttataggt attacatcga agaggatgcc aaggtggctg
     1261 ccgccaataa gtccctcccc gataatgaga agtccctgag cgagaaggat atctttgtga
     1321 ttaacctgag gggctccttc aacgacgacc agaaggatgc cctctactac gatgaagcta
     1381 atagaatttg gagaaagctc gaaaatatca tgcacaacat caaggaattt aggggaaaca
     1441 agacaagaga gtataagaag aaggacgccc ctagactgcc cagaatcctg cccgctggcc
     1501 gtgatgtttc cgccttcagc aaactcatgt atgccctgac catgttcctg gatggcaagg
     1561 agatcaacga cctcctgacc accctgatta ataaattcga taacatccag agcttcctga
     1621 aggtgatgcc tctcatcgga gtcaacgcta agttcgtgga ggaatacgcc tttttcaaag
     1681 actccgccaa gatcgccgat gagctgaggc tgatcaagtc cttcgctaga atgggagaac
     1741 ctattgccga tgccaggagg gccatgtata tcgacgccat ccgtatttta ggaaccaacc
     1801 tgtcctatga tgagctcaag gccctcgccg acaccttttc cctggacgag aacggaaaca
     1861 agctcaagaa aggcaagcac ggcatgagaa atttcattat taataacgtg atcagcaata
     1921 aaaggttcca ctacctgatc agatacggtg atcctgccca cctccatgag atcgccaaaa
     1981 acgaggccgt ggtgaagttc gtgctcggca ggatcgctga catccagaaa aaacagggcc
     2041 agaacggcaa gaaccagatc gacaggtact acgaaacttg tatcggaaag gataagggca
     2101 agagcgtgag cgaaaaggtg gacgctctca caaagatcat caccggaatg aactacgacc
     2161 aattcgacaa gaaaaggagc gtcattgagg acaccggcag ggaaaacgcc gagagggaga
     2221 agtttaaaaa gatcatcagc ctgtacctca ccgtgatcta ccacatcctc aagaatattg
     2281 tcaatatcaa cgccaggtac gtcatcggat tccattgcgt cgagcgtgat gctcaactgt
     2341 acaaggagaa aggctacgac atcaatctca agaaactgga agagaaggga ttcagctccg
     2401 tcaccaagct ctgcgctggc attgatgaaa ctgcccccga taagagaaag gacgtggaaa
     2461 aggagatggc tgaaagagcc aaggagagca ttgacagcct cgagagcgcc aaccccaagc
     2521 tgtatgccaa ttacatcaaa tacagcgacg agaagaaagc cgaggagttc accaggcaga
     2581 ttaacaggga gaaggccaaa accgccctga acgcctacct gaggaacacc aagtggaatg
     2641 tgatcatcag ggaggacctc ctgagaattg acaacaagac atgtaccctg ttcagaaaca
     2701 aggccgtcca cctggaagtg gccaggtatg tccacgccta tatcaacgac attgccgagg
     2761 tcaattccta cttccaactg taccattaca tcatgcagag aattatcatg aatgagaggt
     2821 acgagaaaag cagcggaaag gtgtccgagt acttcgacgc tgtgaatgac gagaagaagt
     2881 acaacgatag gctcctgaaa ctgctgtgtg tgcctttcgg ctactgtatc cccaggttta
     2941 agaacctgag catcgaggcc ctgttcgata ggaacgaggc cgccaagttc gacaaggaga
     3001 aaaagaaggt gtccggcaat tccggatccg gagcggccgc ttacccatac gatgttccag
     3061 attacgctta accgcggatc tggttaccac taaaccagcc tcaagaacac ccgaatggag
     3121 tctctaagct acataatacc aacttacact ttacaaaatg ttgtccccca aaatgtagcc
     3181 attcgtatct gctcctaata aaaagaaagt ttcttcacat tctaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aacccccccc cccccctgca gggccaagtc ggcctctaga ggtacccaat
     3301 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
     3361 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3421 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3481 gaatggcaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3541 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3601 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3661 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3721 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3781 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3841 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3901 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     3961 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatctg
     4021 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4081 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4141 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4201 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4261 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4321 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4381 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4441 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4501 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4561 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4621 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4681 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4741 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4801 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4861 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4921 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4981 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5041 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5101 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5161 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5221 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5281 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5341 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5401 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5461 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5521 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5581 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5641 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5701 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5761 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5821 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5881 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     5941 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     6001 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     6061 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcga
//
