LOCUS       CMV-Cas13X.1-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA       7658 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression vector for encoding a human codon-optimized Cas13X.1 
            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 7658)
  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 7658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
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-Cas13X.1-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA
FEATURES             Location/Qualifiers
     source          1..7658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          139..366
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             387..407
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2730..2750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2760..2894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2797..2816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2851..2870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2901..3149
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2901..2921
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3072..3091
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        3291..3594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3595..3798
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3749..3769
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3827..3836
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3833..4543
                     /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"
     primer_bind     complement(3978..3996)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4560..4784
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5141..5175
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5206..5310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5311..5379,5380..6171)
                     /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(5529..5548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6342..6930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6831..6850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7028..7090
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
       61 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      121 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      181 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      241 cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga
      301 tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt
      361 tttcaggttg gaccggtgcc accatgccca agaagaagcg gaaggtggcc caggtgagca
      421 agcagacctc caagaagagg gagctgagca tcgacgagta ccagggcgcc cggaagtggt
      481 gcttcaccat tgccttcaac aaggccctgg tgaaccggga caagaacgac ggcctgttcg
      541 tggaaagcct gctgagacac gagaagtaca gcaagcacga ctggtacgac gaagataccc
      601 gggccctgat caagtgcagc acccaggccg ccaacgccaa ggctgaagcc ctgcggaact
      661 acttcagtca ctaccggcat agccctggct gcctgacctt caccgccgag gacgaactgc
      721 ggaccatcat ggagagagcc tatgagcggg ccatcttcga gtgcagaaga agagagacag
      781 aggtgatcat cgagtttccc agcctgttcg agggcgaccg gatcaccacc gccggcgtgg
      841 tgtttttcgt gagctttttc gtggaaagaa gagtgctgga tcggctgtat ggagccgtgt
      901 ccggcctgaa gaagaatgag ggacagtaca agctgacccg gaaggccctg agcatgtact
      961 gcctgaagga cagcagattc accaaggcct gggataagcg ggtgctgctg ttcagagaca
     1021 tcctggccca gctgggaaga atccccgccg aggcctacga gtactaccac ggcgagcagg
     1081 gtgataagaa gagagctaac gacaatgagg gcacaaatcc caagcggcac aaggacaagt
     1141 tcatcgaatt tgcactgcac tacctggaag cccagcacag cgagatctgc ttcggcagac
     1201 gccacatcgt gcgggaagag gccggcgccg gcgatgagca caagaagcac cggaccaagg
     1261 gaaaggtggt ggtggacttc agcaagaagg acgaggacca gagctactat atctccaaga
     1321 acaacgtgat cgtgcggatc gacaagaacg ccggccctag aagctaccgg atgggcctga
     1381 acgagctgaa gtacctcgtg ctgctgagcc tgcaggggaa gggcgacgat gccatcgcca
     1441 agctgtacag atacagacag cacgtggaga acatcctgga tgtggtgaag gtgaccgata
     1501 aggataacca cgtgttcctg ccccgcttcg tgctggagca gcacggcatc ggcagaaagg
     1561 ccttcaagca gcggatcgat ggacgggtga agcacgtgcg gggcgtgtgg gagaagaaga
     1621 aggccgccac caatgaaatg accctgcacg agaaggccag agacatcctg cagtacgtga
     1681 acgaaaactg cacccggtcc ttcaaccctg gcgaatacaa cagactgctg gtgtgcctgg
     1741 tgggcaagga cgtggagaac tttcaggccg gcctgaagcg gctgcagctg gccgaaagga
     1801 tcgatggccg ggtgtactcc atcttcgccc agaccagcac catcaatgag atgcaccagg
     1861 tggtgtgcga ccagatcctg aaccggctgt gcagaatcgg cgaccagaag ctgtacgatt
     1921 acgtgggact gggcaagaag gacgaaatcg actacaagca gaaggtggcc tggttcaagg
     1981 agcacatcag catccggaga ggattcctga gaaagaagtt ctggtacgat agcaagaagg
     2041 gattcgcaaa gctggtggag gaacacctgg agtccggcgg cggccagcgc gacgtgggcc
     2101 tggacaagaa gtactaccac atcgacgcca tcggcagatt cgagggcgcc aaccccgccc
     2161 tgtacgagac cctggccaga gatcggctgt gcctcatgat ggcccagtac ttcctgggca
     2221 gcgtgagaaa ggaactgggc aacaagattg tgtggagcaa cgacagcatc gaactgcctg
     2281 tggaaggctc tgtgggaaat gagaagagca tcgtgttctc cgtgtctgac tacggcaagc
     2341 tgtacgtgct ggacgatgcc gaattcctgg gccggatctg cgaatacttc atgccccacg
     2401 aaaagggcaa gatccggtac cacacagtgt acgaaaaggg ctttagagca tacaacgacc
     2461 tgcagaagaa gtgcgtggag gccgtgctgg ctttcgaaga gaaggtggtg aaggccaaga
     2521 agatgagcga gaaggaaggc gcccactaca tcgacttccg ggagatcctg gcccagacca
     2581 tgtgcaagga ggccgaaaag accgcagtga acaaggtgag acgcgccttc ttccaccacc
     2641 acctgaagtt cgtgattgac gagttcggcc tgttcagcga cgtgatgaag aagtacggca
     2701 tcgagaagga atggaagttc cctgtcaagc ccaagaagaa gcggaaggtg tagaagctta
     2761 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2821 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2881 atcatgtctg gatcactagt gagggcctat ttcccatgat tccttcatat ttgcatatac
     2941 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     3001 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     3061 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     3121 tatatatctt gtggaaagga cgaaacaccg gggtcttcga tattcaagcg tcggaagacc
     3181 tgctggagca gcccccgatt tgtggggtga ttacagcttt ttttacgcgt tagttattaa
     3241 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     3301 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     3361 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3421 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3481 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     3541 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     3601 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     3661 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     3721 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     3781 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca ccatggtgag
     3841 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     3901 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     3961 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     4021 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     4081 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     4141 cttcgatgac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     4201 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     4261 gaaaaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     4321 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     4381 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     4441 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     4501 gggccgccac tccaccggcg gcatggacga gctgtacaag taactcttgg cccggatccc
     4561 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4621 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4681 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4741 ggaaaacaat agcaggcatg ctggggatgc ggtgggctct atgggtcgac ggcgcgccct
     4801 agacgaagca gctccagcct acacaatcgc tcaagacgtg taatgctttt attatatatt
     4861 agtcacgata tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     4921 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     4981 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     5041 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     5101 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     5161 actatctttc tagggttaag gatccgcact tttcggggaa atgtgcgcgg aacccctatt
     5221 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5281 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5341 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5401 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5461 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5521 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5581 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5641 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5701 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5761 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5821 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5881 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5941 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6001 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6061 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6121 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6181 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6241 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6301 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6361 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6421 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6481 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6541 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6601 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6661 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6721 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6781 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6841 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6901 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6961 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctctc
     7021 gagttaaccc tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa
     7081 ttgacgcatg tgttttatcg atctgtatat cgaggtttat ttattaattt gaatagatat
     7141 taagttttat tatatttaca cttacatact aataataaat tcaacaaaca atttatttat
     7201 gtttatttat ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac
     7261 ttttaaacat tctctctttt acaaaaataa acttattttg tactttaaaa acagtcatgt
     7321 tgtattataa aataagtaat tagcttaact tatacataat agaaacaaat tatacttatt
     7381 aattaatcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     7441 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
     7501 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     7561 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
     7621 catgacctta tgggactttc ctacttggca gtacatct
//