LOCUS       sequence-380345-        7451 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To express CasRX and a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7451)
  TITLE     CasRx and DjCas13d plasmids
REFERENCE   2  (bases 1 to 7451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAAV-U6-DR30(SapI)-CMV-intron-CasRx-pA
FEATURES             Location/Qualifiers
     source          1..7451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   10..139
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        154..402
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     154..174
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     325..344
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        404..433
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        533..836
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        837..1040
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     991..1011
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1037..1061
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1189..1567
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1650..1670
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1680..4577
                     /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             4587..4607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4617..4643
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4658..4706
                     /note="synthetic polyadenylation signal"
     repeat_region   4723..4863
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4723..4852
                     /label=AAV2 ITR
     rep_origin      4938..5393
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5025..5044)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5235..5256
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5410..5429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5529..5551
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5589..5607)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5675..5779
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5780..5848,5849..6640)
                     /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(5998..6017)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6811..7399
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7300..7319
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
       61 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
      121 tccatcacta ggggttcctg cggccgcact agcgagggcc tatttcccat gattccttca
      181 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      241 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      301 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      361 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaacccct accaactggt
      421 cggggtttga aacggaagag cgagctcttc ttttttttct gtacaacgcg tctagttatt
      481 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      541 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      601 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      661 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      721 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      781 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      841 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      901 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      961 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1021 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     1081 gctgttttga cctccataga agacaccggg accgatccag cctccgcgga ttcgaatccc
     1141 ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc
     1201 tatagagtct ataggcccac aaaaaatgct ttcttctttt aatatacttt tttgtttatc
     1261 ttatttctaa tactttccct aatctctttc tttcagggca ataatgatac aatgtatcat
     1321 gcctctttgc accattctaa agaataacag tgataatttc tgggttaagg caatagcaat
     1381 atttctgcat ataaatattt ctgcatataa attgtaactg atgtaagagg tttcatattg
     1441 ctaatagcag ctacaatcca gctaccattc tgcttttatt ttatggttgg gataaggctg
     1501 gattattctg agtccaagct aggccctttt gctaatcatg ttcatacctc ttatcttcct
     1561 cccacagctc ctgggcaacg tgctggtctg tgtgctggcc catcactttg gcaaagaatt
     1621 gggattcgaa catcgatgcc accatgagcc ccaagaagaa gagaaaggtg gaggccagca
     1681 tcgaaaaaaa aaagtccttc gccaagggca tgggcgtgaa gtccacactc gtgtccggct
     1741 ccaaagtgta catgacaacc ttcgccgaag gcagcgacgc caggctggaa aagatcgtgg
     1801 agggcgacag catcaggagc gtgaatgagg gcgaggcctt cagcgctgaa atggccgata
     1861 aaaacgccgg ctataagatc ggcaacgcca aattcagcca tcctaagggc tacgccgtgg
     1921 tggctaacaa ccctctgtat acaggacccg tccagcagga tatgctcggc ctgaaggaaa
     1981 ctctggaaaa gaggtacttc ggcgagagcg ctgatggcaa tgacaatatt tgtatccagg
     2041 tgatccataa catcctggac attgaaaaaa tcctcgccga atacattacc aacgccgcct
     2101 acgccgtcaa caatatctcc ggcctggata aggacattat tggattcggc aagttctcca
     2161 cagtgtatac ctacgacgaa ttcaaagacc ccgagcacca tagggccgct ttcaacaata
     2221 acgataagct catcaacgcc atcaaggccc agtatgacga gttcgacaac ttcctcgata
     2281 accccagact cggctatttc ggccaggcct ttttcagcaa ggagggcaga aattacatca
     2341 tcaattacgg caacgaatgc tatgacattc tggccctcct gagcggactg aggcactggg
     2401 tggtccataa caacgaagaa gagtccagga tctccaggac ctggctctac aacctcgata
     2461 agaacctcga caacgaatac atctccaccc tcaactacct ctacgacagg atcaccaatg
     2521 agctgaccaa ctccttctcc aagaactccg ccgccaacgt gaactatatt gccgaaactc
     2581 tgggaatcaa ccctgccgaa ttcgccgaac aatatttcag attcagcatt atgaaagagc
     2641 agaaaaacct cggattcaat atcaccaagc tcagggaagt gatgctggac aggaaggata
     2701 tgtccgagat caggaaaaat cataaggtgt tcgactccat caggaccaag gtctacacca
     2761 tgatggactt tgtgatttat aggtattaca tcgaagagga tgccaaggtg gctgccgcca
     2821 ataagtccct ccccgataat gagaagtccc tgagcgagaa ggatatcttt gtgattaacc
     2881 tgaggggctc cttcaacgac gaccagaagg atgccctcta ctacgatgaa gctaatagaa
     2941 tttggagaaa gctcgaaaat atcatgcaca acatcaagga atttagggga aacaagacaa
     3001 gagagtataa gaagaaggac gcccctagac tgcccagaat cctgcccgct ggccgtgatg
     3061 tttccgcctt cagcaaactc atgtatgccc tgaccatgtt cctggatggc aaggagatca
     3121 acgacctcct gaccaccctg attaataaat tcgataacat ccagagcttc ctgaaggtga
     3181 tgcctctcat cggagtcaac gctaagttcg tggaggaata cgcctttttc aaagactccg
     3241 ccaagatcgc cgatgagctg aggctgatca agtccttcgc tagaatggga gaacctattg
     3301 ccgatgccag gagggccatg tatatcgacg ccatccgtat tttaggaacc aacctgtcct
     3361 atgatgagct caaggccctc gccgacacct tttccctgga cgagaacgga aacaagctca
     3421 agaaaggcaa gcacggcatg agaaatttca ttattaataa cgtgatcagc aataaaaggt
     3481 tccactacct gatcagatac ggtgatcctg cccacctcca tgagatcgcc aaaaacgagg
     3541 ccgtggtgaa gttcgtgctc ggcaggatcg ctgacatcca gaaaaaacag ggccagaacg
     3601 gcaagaacca gatcgacagg tactacgaaa cttgtatcgg aaaggataag ggcaagagcg
     3661 tgagcgaaaa ggtggacgct ctcacaaaga tcatcaccgg aatgaactac gaccaattcg
     3721 acaagaaaag gagcgtcatt gaggacaccg gcagggaaaa cgccgagagg gagaagttta
     3781 aaaagatcat cagcctgtac ctcaccgtga tctaccacat cctcaagaat attgtcaata
     3841 tcaacgccag gtacgtcatc ggattccatt gcgtcgagcg tgatgctcaa ctgtacaagg
     3901 agaaaggcta cgacatcaat ctcaagaaac tggaagagaa gggattcagc tccgtcacca
     3961 agctctgcgc tggcattgat gaaactgccc ccgataagag aaaggacgtg gaaaaggaga
     4021 tggctgaaag agccaaggag agcattgaca gcctcgagag cgccaacccc aagctgtatg
     4081 ccaattacat caaatacagc gacgagaaga aagccgagga gttcaccagg cagattaaca
     4141 gggagaaggc caaaaccgcc ctgaacgcct acctgaggaa caccaagtgg aatgtgatca
     4201 tcagggagga cctcctgaga attgacaaca agacatgtac cctgttcaga aacaaggccg
     4261 tccacctgga agtggccagg tatgtccacg cctatatcaa cgacattgcc gaggtcaatt
     4321 cctacttcca actgtaccat tacatcatgc agagaattat catgaatgag aggtacgaga
     4381 aaagcagcgg aaaggtgtcc gagtacttcg acgctgtgaa tgacgagaag aagtacaacg
     4441 ataggctcct gaaactgctg tgtgtgcctt tcggctactg tatccccagg tttaagaacc
     4501 tgagcatcga ggccctgttc gataggaacg aggccgccaa gttcgacaag gagaaaaaga
     4561 aggtgtccgg caattccgga tccggaccta agaaaaagag gaaggtggcg gccgcttacc
     4621 catacgatgt tccagattac gcttaggcta ggaattcaat aaaagatctt tattttcatt
     4681 agatctgtgt gttggttttt tgtgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     4741 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4801 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4861 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     4921 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4981 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5041 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5101 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5161 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5221 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5281 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5341 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5401 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5461 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5521 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5581 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5641 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5701 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5761 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5821 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5881 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5941 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6001 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6061 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6121 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6181 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6241 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6301 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6361 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6421 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6481 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6541 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6601 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6661 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6721 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6781 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6841 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6901 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6961 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7021 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7081 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7141 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7201 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7261 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7321 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7381 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7441 ctggcctttt g
//
