LOCUS       sequence-337353-        8703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for encoding a human codon-optimized 
            minidCas13X.1-RESCUE-S 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 8703)
  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 8703)
  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-minidCas13X.1-RESCUE-S-SV40pA_U6-BbsI-DR_CMV-mCherry-P2A-Puro-BG
            HpA
FEATURES             Location/Qualifiers
     source          1..8703
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   25..87
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        396..681
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        683..960
                     /label=chicken beta-actin promoter
     intron          961..1188
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1209..1229
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2562..2582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3810..3944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3847..3866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3901..3920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3951..4199
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3951..3971
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4122..4141
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        4341..4644
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4645..4848
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4799..4819
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4877..4886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4883..5590
                     /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(5028..5046)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             5600..5656
                     /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             5657..6253
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     6150..6170
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6260..6484
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6841..6875
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        6906..7010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7011..7079,7080..7871)
                     /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(7229..7248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8042..8630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8531..8550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatcccctga ttctctcgag ttaaccctag aaagataatc atattgtgac gtacgttaaa
       61 gataatcatg cgtaaaattg acgcatgtgt tttatcgatc tgtatatcga ggtttattta
      121 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
      181 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
      241 ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact tattttgtac
      301 tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat acataataga
      361 aacaaattat acttattaat taatctagag gtacccgtta cataacttac ggtaaatggc
      421 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg
      481 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      541 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      601 tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      661 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      721 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      781 atgggggcgg gggggggggg ggggcgcgcg ccrggsgggg sggggsgggg sgrggggsgg
      841 ggsggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      901 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      961 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1021 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1081 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     1141 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     1201 ccaccatgcc taagaaaaag cgtaaggtcc tgagcatgta ctgcctgaag gacagcagat
     1261 tcaccaaggc ctgggataag cgggtgctgc tgttcagaga catcctggcc cagctgggaa
     1321 gaatccccgc cgaggcctac gagtactacc acggcgagca gggtgataag aagagagcta
     1381 acgacaatga gggcacaaat cccaagcggc acaaggacaa gttcatcgaa tttgcactgc
     1441 actacctgga agcccagcac agcgagatct gcttcggcag acgccacatc gtgcgggaag
     1501 aggccggcgc cggcgatgag cacaagaagc accggaccaa gggaaaggtg gtggtggact
     1561 tcagcaagaa ggacgaggac cagagctact atatctccaa gaacaacgtg atcgtgcgga
     1621 tcgacaagaa cgccggccct agaagctacc ggatgggcct gaacgagctg aagtacctcg
     1681 tgctgctgag cctgcagggg aagggcgacg atgccatcgc caagctgtac agatacagac
     1741 agcacgtgga gaacatcctg gatgtggtga aggtgaccga taaggataac cacgtgttcc
     1801 tgccccgctt cgtgctggag cagcacggca tcggcagaaa ggccttcaag cagcggatcg
     1861 atggacgggt gaagcacgtg cggggcgtgt gggagaagaa gaaggccgcc accaatgaaa
     1921 tgaccctgca cgagaaggcc agagacatcc tgcagtacgt gaacgaaaac tgcacccggt
     1981 ccttcaaccc tggcgaatac aacagactgc tggtgtgcct ggtgggcaag gacgtggaga
     2041 actttcaggc cggcctgaag cggctgcagc tggccgaaag gatcgatggc cgggtgtact
     2101 ccatcttcgc ccagaccagc accatcaatg agatgcacca ggtggtgtgc gaccagatcc
     2161 tgaaccggct gtgcagaatc ggcgaccaga agctgtacga ttacgtggga ctgggcaaga
     2221 aggacgaaat cgactacaag cagaaggtgg cctggttcaa ggagcacatc agcatccgga
     2281 gaggattcct gagaaagaag ttctggtacg atagcaagaa gggattcgca aagctggtgg
     2341 aggaacacct ggagtccggc ggcggccagc gcgacgtggg cctggacaag aagtactacc
     2401 acatcgacgc catcggcaga ttcgagggcg ccaaccccgc cctgtacgag accctggcca
     2461 gagatcggct gtgcctcatg atggcccagt acttcctggg cagcgtgaga aaggaactgg
     2521 gcaacaagat tgtgtggagc aacgacagca tcgaactgcc tcccaagaag aagcggaagg
     2581 tgggtggagg cggaggttct gggggaggag gtagtggcgg tggtggttca ggaggcggcg
     2641 gaagccagct gcatttaccg caggttttag ctgacgctgt ctcacgcctg gtcataggta
     2701 agtttggtga cctgaccgac aacttctcct cccctcacgc tcgcagaata ggtctggctg
     2761 gagtcgtcat gacaacaggc acagatgtta aagatgccaa ggtgatatgt gtttctacag
     2821 gagcaaaatg tattaatggt gaatacctaa gtgatcgtgg ccttgcatta aatgactgcc
     2881 atgcagaaat agtatctcgg agatccttgc tcagatttct ttatacacaa cttgagcttt
     2941 acttaaataa cgaggatgat caaaaaagat ccatctttca gaaatcagag cgaggggggt
     3001 ttaggctgaa ggagaatata cagtttcatc tgtacatcag cacctctccc tgtggagatg
     3061 ccagaatctt ctcaccacat gaggcaatcc tggaagaacc agcagataga cacccaaatc
     3121 gtaaagcaag aggacagcta cggaccaaaa tagaggctgg tcaggggacg attccagtgc
     3181 gcaacaatgc gagcatccaa acgtgggacg gggtgctgca aggggagcgg ctgctcacca
     3241 tgtcctgcag tgacaagatt gcacgctgga acgtggtggg catccaggga tcactgctca
     3301 gcattttcgt ggagcccatt tacttctcga gcatcatcct gggcagcctt taccacgggg
     3361 accacctttc cagggccatg taccagcgga tctccaacat agaggacctg ccacctctct
     3421 acaccctcaa caagcctttg ctcacaggca tcagcaatgc agaagcacgg cagccaggga
     3481 aggcccccat attcagtgtc aactggacgg taggcgactc cgctattgag gtcatcaacg
     3541 ccacgactgg gaagggagag ctgggccgcg cgtcccgcct gtgtaagcac gcgttgtact
     3601 gtcgctggat gcgtgtgcac ggcaaggttc cctcccactt actacgctcc aagattacca
     3661 agcccaacgt gtaccatgag acaaagctgg cggcaaagga gtaccaggcc gccaaggcgc
     3721 gtctgttcac agccttcatc aaggcggggc tgggggcctg ggtggagaag cccaccgagc
     3781 aggaccagtt ctcactcacg taaaagctta acttgtttat tgcagcttat aatggttaca
     3841 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3901 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcactagt gagggcctat
     3961 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     4021 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     4081 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     4141 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     4201 gggtcttcga tattcaagcg tcggaagacc tgctggagca gcccccgatt tgtggggtga
     4261 ttacagcttt ttttacgcgt tagttattaa tagtaatcaa ttacggggtc attagttcat
     4321 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     4381 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     4441 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     4501 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     4561 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     4621 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     4681 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     4741 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     4801 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     4861 cgtcagatcc gctagcgcca ccatggtgag caagggcgag gaggataaca tggccatcat
     4921 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     4981 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     5041 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     5101 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     5161 cgagggcttc aagtgggagc gcgtgatgaa cttcgatgac ggcggcgtgg tgaccgtgac
     5221 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     5281 cttcccctcc gacggccccg taatgcagaa gaaaaccatg ggctgggagg cctcctccga
     5341 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     5401 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     5461 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     5521 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     5581 gctgtacaag ggtagtgggg caacaaactt ctctctgctg aaacaagccg gagatgtcga
     5641 agagaatcct ggaccgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     5701 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     5761 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     5821 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     5881 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     5941 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     6001 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     6061 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     6121 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     6181 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     6241 gcccggtgcc tgacttaagc tgtgccttct agttgccagc catctgttgt ttgcccctcc
     6301 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     6361 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     6421 gacagcaagg gggaggattg ggaaaacaat agcaggcatg ctggggatgc ggtgggctct
     6481 atgggtcgac ggcgcgccct agacgaagca gctccagcct acacaatcgc tcaagacgtg
     6541 taatgctttt attatatatt agtcacgata tctataacaa gaaaatatat atataataag
     6601 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     6661 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     6721 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     6781 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     6841 catgcgtcaa ttttacgcag actatctttc tagggttaag gatccgcact tttcggggaa
     6901 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6961 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     7021 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     7081 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     7141 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     7201 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     7261 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     7321 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     7381 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7441 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7501 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7561 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7621 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7681 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7741 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7801 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7861 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7921 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7981 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     8041 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     8101 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     8161 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     8221 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     8281 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     8341 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     8401 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8461 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8521 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8581 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8641 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8701 cgt
//
