LOCUS Exported 8703 bp ds-DNA circular SYN 12-JUL-2021 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 CMV-minidCas13X.1-RESCUE-S-SV40pA_U6-BbsI-DR_CMV-mCherry-... 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 Jul 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8703 /organism="synthetic DNA construct" /mol_type="other DNA" 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..4191 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 7011..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 //