LOCUS CMV-RfxCas13d-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA 8250 bp DNA circular SYN 25-AUG-2024
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 Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.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"
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 5759..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
//
