LOCUS       sequence-337345-        8250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for encoding a human codon-optimized RfxCas13d 
            driven by CMV promoter,mCherry driven by CMV promoter and U6-driven 
            crRNAs cloning site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8250)
  AUTHORS   Xu C, Zhou Y, Xiao Q, He B, Geng G, Wang Z, Cao B, Dong X, Bai W, 
            Wang Y, Wang X, Zhou D, Yuan T, Huo X, Lai J, Yang H
  TITLE     Programmable RNA editing with compact CRISPR-Cas13 systems from 
            uncultivated microbes.
  JOURNAL   Nat Methods. 2021 May;18(5):499-506. doi: 
            10.1038/s41592-021-01124-4. Epub 2021 May 3.
  PUBMED    33941935
REFERENCE   2  (bases 1 to 8250)
  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: 
            "10.1038/s41592-021-01124-4"; journalName: "Nat Methods"; date: 
            "2021-05"; volume: "18"; issue: "5"; pages: "499-506"
COMMENT     Sequence Label: CMV-RfxCas13d-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA
FEATURES             Location/Qualifiers
     source          1..8250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             224..244
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             254..3151
                     /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             3161..3187
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3188..3208
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3221..3355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3258..3277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3312..3331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3362..3610
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3362..3382
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3533..3552
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3612..3647
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     enhancer        3739..4042
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4043..4246
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4197..4217
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4275..4284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4281..4991
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     DDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    5008..5232
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5589..5623
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5654..5758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5759..5827,5828..6619)
                     /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(5977..5996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6790..7378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7279..7298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7476..7538
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        7847..8132
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
       61 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
      121 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
      181 tgcctgaaat cacttttttt caggttggac cggtgccacc atgcctaaaa agaaaagaaa
      241 ggtgggttct ggtatcgaga agaagaagag cttcgccaag ggcatgggag tgaagagcac
      301 cctggtgtcc ggctctaagg tgtacatgac cacatttgct gagggaagcg acgccaggct
      361 ggagaagatc gtggagggcg atagcatcag atccgtgaac gagggagagg ctttcagcgc
      421 cgagatggct gacaagaacg ctggctacaa gatcggaaac gccaagtttt cccacccaaa
      481 gggctacgcc gtggtggcta acaacccact gtacaccgga ccagtgcagc aggacatgct
      541 gggactgaag gagacactgg agaagaggta cttcggcgag tccgccgacg gaaacgataa
      601 catctgcatc caggtcatcc acaacatcct ggatatcgag aagatcctgg ctgagtacat
      661 cacaaacgcc gcttacgccg tgaacaacat ctccggcctg gacaaggata tcatcggctt
      721 cggaaagttt tctaccgtgt acacatacga cgagttcaag gatccagagc accaccgggc
      781 cgcttttaac aacaacgaca agctgatcaa cgccatcaag gctcagtacg acgagttcga
      841 taactttctg gataacccca ggctgggcta cttcggacag gctttctttt ctaaggaggg
      901 cagaaactac atcatcaact acggaaacga gtgttacgac atcctggccc tgctgagcgg
      961 actgaggcac tgggtggtgc acaacaacga ggaggagtct cggatcagcc gcacctggct
     1021 gtacaacctg gacaagaacc tggataacga gtacatctcc acactgaact acctgtacga
     1081 caggatcacc aacgagctga caaacagctt ctccaagaac tctgccgcta acgtgaacta
     1141 catcgctgag accctgggca tcaacccagc tgagttcgct gagcagtact tcagattttc
     1201 catcatgaag gagcagaaga acctgggctt caacatcaca aagctgagag aagtgatgct
     1261 ggacagaaag gatatgtccg agatcaggaa gaaccacaag gtgttcgatt ctatcagaac
     1321 caaggtgtac acaatgatgg actttgtgat ctacaggtac tacatcgagg aggatgccaa
     1381 ggtggccgct gccaacaaga gcctgcccga caacgagaag tctctgagcg agaaggatat
     1441 cttcgtgatc aacctgagag gctcctttaa cgacgatcag aaggacgctc tgtactacga
     1501 tgaggccaac aggatctgga gaaagctgga gaacatcatg cacaacatca aggagttccg
     1561 gggaaacaag acccgcgagt acaagaagaa ggacgctcca aggctgccta ggatcctgcc
     1621 tgctggaagg gacgtgagcg ccttcagcaa gctgatgtac gccctgacaa tgtttctgga
     1681 cggaaaggag atcaacgatc tgctgaccac actgatcaac aagttcgaca acatccagtc
     1741 ttttctgaaa gtgatgcctc tgatcggcgt gaacgctaag ttcgtggagg agtacgcctt
     1801 ctttaaggac agcgccaaga tcgctgatga gctgcggctg atcaagtcct ttgccaggat
     1861 gggagagcca atcgctgacg ctaggagagc tatgtacatc gatgccatcc ggatcctggg
     1921 aaccaacctg tcttacgacg agctgaaggc tctggccgac accttcagcc tggatgagaa
     1981 cggcaacaag ctgaagaagg gcaagcacgg aatgcgcaac ttcatcatca acaacgtgat
     2041 cagcaacaag cggtttcact acctgatcag atacggcgac ccagctcacc tgcacgagat
     2101 cgctaagaac gaggccgtgg tgaagttcgt gctgggacgg atcgccgata tccagaagaa
     2161 gcagggccag aacggaaaga accagatcga ccgctactac gagacctgca tcggcaagga
     2221 taagggaaag tccgtgtctg agaaggtgga cgctctgacc aagatcatca caggcatgaa
     2281 ctacgaccag ttcgataaga agagatctgt gatcgaggac accggaaggg agaacgccga
     2341 gagagagaag tttaagaaga tcatcagcct gtacctgaca gtgatctacc acatcctgaa
     2401 gaacatcgtg aacatcaacg ctagatacgt gatcggcttc cactgcgtgg agcgcgatgc
     2461 ccagctgtac aaggagaagg gatacgacat caacctgaag aagctggagg agaagggctt
     2521 tagctccgtg accaagctgt gcgctggaat cgacgagaca gcccccgaca agaggaagga
     2581 tgtggagaag gagatggccg agagagctaa ggagagcatc gactccctgg agtctgctaa
     2641 ccctaagctg tacgccaact acatcaagta ctccgatgag aagaaggccg aggagttcac
     2701 caggcagatc aacagagaga aggccaagac cgctctgaac gcctacctga ggaacacaaa
     2761 gtggaacgtg atcatccggg aggacctgct gcgcatcgat aacaagacct gtacactgtt
     2821 ccggaacaag gctgtgcacc tggaggtggc tcgctacgtg cacgcctaca tcaacgacat
     2881 cgccgaggtg aactcctact ttcagctgta ccactacatc atgcagagga tcatcatgaa
     2941 cgagagatac gagaagtcta gcggcaaggt gtctgagtac ttcgacgccg tgaacgatga
     3001 gaagaagtac aacgatagac tgctgaagct gctgtgcgtg cctttcggat actgtatccc
     3061 acggtttaag aacctgagca tcgaggccct gttcgaccgc aacgaggctg ccaagtttga
     3121 taaggagaag aagaaggtga gcggcaactc cggttctggt tacccatacg acgtaccaga
     3181 ttacgctccc aagaagaagc gcaaggtggg ataaaagctt aacttgttta ttgcagctta
     3241 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3301 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcactag
     3361 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     3421 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     3481 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     3541 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     3601 acgaaacacc gcaagtaaac ccctaccaac tggtcggggt ttgaaacggg tcttcgagaa
     3661 gacctttttt ttacgcgtta gttattaata gtaatcaatt acggggtcat tagttcatag
     3721 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     3781 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     3841 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     3901 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3961 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     4021 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     4081 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     4141 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     4201 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     4261 tcagatccgc tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca
     4321 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     4381 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     4441 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     4501 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     4561 agggcttcaa gtgggagcgc gtgatgaact tcgatgacgg cggcgtggtg accgtgaccc
     4621 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     4681 tcccctccga cggccccgta atgcagaaga aaaccatggg ctgggaggcc tcctccgagc
     4741 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     4801 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     4861 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     4921 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     4981 tgtacaagta actcttggcc cggatccctg tgccttctag ttgccagcca tctgttgttt
     5041 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5101 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5161 tggggcagga cagcaagggg gaggattggg aaaacaatag caggcatgct ggggatgcgg
     5221 tgggctctat gggtcgacgg cgcgccctag acgaagcagc tccagcctac acaatcgctc
     5281 aagacgtgta atgcttttat tatatattag tcacgatatc tataacaaga aaatatatat
     5341 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
     5401 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
     5461 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
     5521 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
     5581 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaagga tccgcacttt
     5641 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5701 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5761 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5821 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5881 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5941 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6001 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6061 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6121 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6181 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6241 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6301 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6361 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6421 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6481 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6541 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6601 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6661 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6721 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6781 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6841 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6901 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6961 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7021 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7081 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7141 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7201 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7261 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7321 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7381 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7441 ctttcctgcg ttatcccctg attctctcga gttaacccta gaaagataat catattgtga
     7501 cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcgat ctgtatatcg
     7561 aggtttattt attaatttga atagatatta agttttatta tatttacact tacatactaa
     7621 taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa aacaaaaact
     7681 caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac aaaaataaac
     7741 ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta gcttaactta
     7801 tacataatag aaacaaatta tacttattaa ttaatctaga ggtacccgtt acataactta
     7861 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa
     7921 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     7981 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     8041 aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt
     8101 acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca
     8161 ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat
     8221 tttgtgcagc gatgggggcg gggggggggg
//
