LOCUS       sequence-468663-        7457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid to carry out IVT of High-fidelity RfxCas13d (human 
            codon-optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7457)
  AUTHORS   Moreno-Sanchez I, Hernandez-Huertas L, Nahon-Cano D, Martinez-Garcia
            PM, Treichel AJ, Gomez-Marin C, Tomas-Gallardo L, da Silva Pescador 
            G, Kushawah G, Egidy R, Perera A, Diaz-Moscoso A, Cano-Ruiz A, 
            Walker JA 2nd, Munoz MJ, Holden K, Galceran J, Nieto MA, Bazzini AA,
            Moreno-Mateos MA
  TITLE     Enhanced RNA-targeting CRISPR-Cas technology in zebrafish.
  JOURNAL   Nat Commun. 2025 Mar 16;16(1):2591. doi: 10.1038/s41467-025-57792-9.
  PUBMED    40091120
REFERENCE   2  (bases 1 to 7457)
  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: "Nat 
            Commun."; date: "2025-03-16"; volume: "16(1):2591. doi"; pages: " 
            10.1038/s41467-025-57792-9"
COMMENT     Sequence Label: pT3TS-Hf-RfxCas13d-HA
FEATURES             Location/Qualifiers
     source          1..7457
                     /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
                     RYFGESADGNDNICIQVIHNILDIEKILVEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     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"
     primer_bind     complement(3307..3326)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3308..3326)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3333..3350)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3333..3349)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3342..3364)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3491..3946)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3628..3649)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3840..3859
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3973..4077
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4078..4146,4147..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
                     LIKH"
     primer_bind     complement(4296..4315)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6447..7035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6936..6955
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7189..7206
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7323..7344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7359..7364,7365..7382,7383..7389)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7397..7413
                     /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     7402..7424
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7421..7437
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7421..7437
                     /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 cgtcgaatac 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 ttctccccca caaaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaccccccc ccccccccct gcagggccaa gtcggcctct agaggtaccc
     3301 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac
     3361 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     3421 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     3481 ggcgaatggc aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3541 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3601 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3661 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3721 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3781 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3841 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3901 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     3961 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4021 ctgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4081 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4141 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4201 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4261 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4321 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4381 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4441 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4501 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4561 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4621 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4681 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4741 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4801 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4861 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4921 ctgattaagc actgagagat cccctcataa tttccccaaa gcgtaaccat gtgtgaataa
     4981 attttgagct agtagggttg cagccacgag taagtcttcc cttgttattg tgtagccaga
     5041 atgccgcaaa acttccatgc ctaagcgaac tgttgagagt acgtttcgat ttctgactgt
     5101 gttagcctgg aagtgcttgt cccaaccttg tttctgagca tgaacgcccg caagccaaca
     5161 tgttagttga agcatcaggg cgattagcag catgatatca aaacgctctg agctgctcgt
     5221 tcggctatgg cgtaggccta gtccgtaggc aggacttttc aagtctcgga aggtttcttc
     5281 aatctgcatt cgcttcgaat agatattaac aagttgtttg ggtgttcgaa tttcaacagg
     5341 taagttagtt gctagaatcc atggctcctt tgccgacgct gagtagattt taggtgacgg
     5401 gtggtgacaa tgagtccgtg tcgagcgctg attttttcgg cctttagagc gagatttata
     5461 caatagaatt tggcatgaga ttggattgct tttagtcagc ctcttatagc ctaaagtctt
     5521 tgagtgacta gatgacatat catgtaagtt gctgataggt ttccagtttt ccgctcctag
     5581 gtctgcatat tgtacttttc ctcttactcg acttaaccag taccaaccca gcttctcaac
     5641 ggatttatac catggcactt taaagccagc atcactgaca atgagcggtg tggtgttact
     5701 cggtagaatg ctcgcaaggt cggctagaaa ttggtcatga gctttctttg aacattgctc
     5761 tgaaagcggg aacgctttct cataaagagt aacagaacga ccgtgtagtg cgactgaagc
     5821 tcgcaatacc ataagccgtt tttgctcacg gatatcagac cagtcaacaa gtacaatggg
     5881 catcgtattg cccgaacaga taaagctagc atgccaacgg tatacagcga gtcgctcttt
     5941 gtggaggtga cgattaccta acaatcggtc gattcgtttg atgttatgtt ttgttctcgc
     6001 tttggttggc aggttacggc caagttcggt aagagtgaga gttttacagt caagtaaggc
     6061 gtggcaagcc aacgttaagc tgttgagtcg ttttaagtgt aattcggggc agaattggta
     6121 aagagagtcg tgtaaaatat cgagttcgca cattttgttg tctgattatt gatttttggc
     6181 gaaaccattt gatcatatga caagatgtgt atctacctta acttaatgat tttgataaaa
     6241 atcattaggg gattcatcag gattaagcat tggtaactgt cagaccaagt ttactcatat
     6301 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6361 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6421 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6481 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6541 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6601 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6661 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6721 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6781 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6841 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6901 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6961 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7021 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7081 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7141 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7201 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7261 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7321 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7381 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     7441 gattacgcca agctcga
//
