LOCUS       sequence-381959-        7133 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To express CasRx  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7133)
  TITLE     CasRx and DjCas13d plasmids
REFERENCE   2  (bases 1 to 7133)
  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-CMV-intron-CasRx-pA
FEATURES             Location/Qualifiers
     source          1..7133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   8..137
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        213..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        517..720
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     671..691
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     717..741
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          869..1247
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1330..1350
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1360..4257
                     /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             4267..4287
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4297..4323
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4338..4386
                     /note="synthetic polyadenylation signal"
     repeat_region   4403..4543
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4403..4532
                     /label=AAV2 ITR
     rep_origin      4618..5073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4705..4724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4915..4936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5090..5109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5209..5231
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5269..5287)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5355..5459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5460..5528,5529..6320)
                     /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(5678..5697)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6491..7079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6980..6999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
       61 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      121 catcactagg ggttcctgcg gccgcacgcg tctagttatt aatagtaatc aattacgggg
      181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      721 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
      781 agacaccggg accgatccag cctccgcgga ttcgaatccc ggccgggaac ggtgcattgg
      841 aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc tatagagtct ataggcccac
      901 aaaaaatgct ttcttctttt aatatacttt tttgtttatc ttatttctaa tactttccct
      961 aatctctttc tttcagggca ataatgatac aatgtatcat gcctctttgc accattctaa
     1021 agaataacag tgataatttc tgggttaagg caatagcaat atttctgcat ataaatattt
     1081 ctgcatataa attgtaactg atgtaagagg tttcatattg ctaatagcag ctacaatcca
     1141 gctaccattc tgcttttatt ttatggttgg gataaggctg gattattctg agtccaagct
     1201 aggccctttt gctaatcatg ttcatacctc ttatcttcct cccacagctc ctgggcaacg
     1261 tgctggtctg tgtgctggcc catcactttg gcaaagaatt gggattcgaa catcgatgcc
     1321 accatgagcc ccaagaagaa gagaaaggtg gaggccagca tcgaaaaaaa aaagtccttc
     1381 gccaagggca tgggcgtgaa gtccacactc gtgtccggct ccaaagtgta catgacaacc
     1441 ttcgccgaag gcagcgacgc caggctggaa aagatcgtgg agggcgacag catcaggagc
     1501 gtgaatgagg gcgaggcctt cagcgctgaa atggccgata aaaacgccgg ctataagatc
     1561 ggcaacgcca aattcagcca tcctaagggc tacgccgtgg tggctaacaa ccctctgtat
     1621 acaggacccg tccagcagga tatgctcggc ctgaaggaaa ctctggaaaa gaggtacttc
     1681 ggcgagagcg ctgatggcaa tgacaatatt tgtatccagg tgatccataa catcctggac
     1741 attgaaaaaa tcctcgccga atacattacc aacgccgcct acgccgtcaa caatatctcc
     1801 ggcctggata aggacattat tggattcggc aagttctcca cagtgtatac ctacgacgaa
     1861 ttcaaagacc ccgagcacca tagggccgct ttcaacaata acgataagct catcaacgcc
     1921 atcaaggccc agtatgacga gttcgacaac ttcctcgata accccagact cggctatttc
     1981 ggccaggcct ttttcagcaa ggagggcaga aattacatca tcaattacgg caacgaatgc
     2041 tatgacattc tggccctcct gagcggactg aggcactggg tggtccataa caacgaagaa
     2101 gagtccagga tctccaggac ctggctctac aacctcgata agaacctcga caacgaatac
     2161 atctccaccc tcaactacct ctacgacagg atcaccaatg agctgaccaa ctccttctcc
     2221 aagaactccg ccgccaacgt gaactatatt gccgaaactc tgggaatcaa ccctgccgaa
     2281 ttcgccgaac aatatttcag attcagcatt atgaaagagc agaaaaacct cggattcaat
     2341 atcaccaagc tcagggaagt gatgctggac aggaaggata tgtccgagat caggaaaaat
     2401 cataaggtgt tcgactccat caggaccaag gtctacacca tgatggactt tgtgatttat
     2461 aggtattaca tcgaagagga tgccaaggtg gctgccgcca ataagtccct ccccgataat
     2521 gagaagtccc tgagcgagaa ggatatcttt gtgattaacc tgaggggctc cttcaacgac
     2581 gaccagaagg atgccctcta ctacgatgaa gctaatagaa tttggagaaa gctcgaaaat
     2641 atcatgcaca acatcaagga atttagggga aacaagacaa gagagtataa gaagaaggac
     2701 gcccctagac tgcccagaat cctgcccgct ggccgtgatg tttccgcctt cagcaaactc
     2761 atgtatgccc tgaccatgtt cctggatggc aaggagatca acgacctcct gaccaccctg
     2821 attaataaat tcgataacat ccagagcttc ctgaaggtga tgcctctcat cggagtcaac
     2881 gctaagttcg tggaggaata cgcctttttc aaagactccg ccaagatcgc cgatgagctg
     2941 aggctgatca agtccttcgc tagaatggga gaacctattg ccgatgccag gagggccatg
     3001 tatatcgacg ccatccgtat tttaggaacc aacctgtcct atgatgagct caaggccctc
     3061 gccgacacct tttccctgga cgagaacgga aacaagctca agaaaggcaa gcacggcatg
     3121 agaaatttca ttattaataa cgtgatcagc aataaaaggt tccactacct gatcagatac
     3181 ggtgatcctg cccacctcca tgagatcgcc aaaaacgagg ccgtggtgaa gttcgtgctc
     3241 ggcaggatcg ctgacatcca gaaaaaacag ggccagaacg gcaagaacca gatcgacagg
     3301 tactacgaaa cttgtatcgg aaaggataag ggcaagagcg tgagcgaaaa ggtggacgct
     3361 ctcacaaaga tcatcaccgg aatgaactac gaccaattcg acaagaaaag gagcgtcatt
     3421 gaggacaccg gcagggaaaa cgccgagagg gagaagttta aaaagatcat cagcctgtac
     3481 ctcaccgtga tctaccacat cctcaagaat attgtcaata tcaacgccag gtacgtcatc
     3541 ggattccatt gcgtcgagcg tgatgctcaa ctgtacaagg agaaaggcta cgacatcaat
     3601 ctcaagaaac tggaagagaa gggattcagc tccgtcacca agctctgcgc tggcattgat
     3661 gaaactgccc ccgataagag aaaggacgtg gaaaaggaga tggctgaaag agccaaggag
     3721 agcattgaca gcctcgagag cgccaacccc aagctgtatg ccaattacat caaatacagc
     3781 gacgagaaga aagccgagga gttcaccagg cagattaaca gggagaaggc caaaaccgcc
     3841 ctgaacgcct acctgaggaa caccaagtgg aatgtgatca tcagggagga cctcctgaga
     3901 attgacaaca agacatgtac cctgttcaga aacaaggccg tccacctgga agtggccagg
     3961 tatgtccacg cctatatcaa cgacattgcc gaggtcaatt cctacttcca actgtaccat
     4021 tacatcatgc agagaattat catgaatgag aggtacgaga aaagcagcgg aaaggtgtcc
     4081 gagtacttcg acgctgtgaa tgacgagaag aagtacaacg ataggctcct gaaactgctg
     4141 tgtgtgcctt tcggctactg tatccccagg tttaagaacc tgagcatcga ggccctgttc
     4201 gataggaacg aggccgccaa gttcgacaag gagaaaaaga aggtgtccgg caattccgga
     4261 tccggaccta agaaaaagag gaaggtggcg gccgcttacc catacgatgt tccagattac
     4321 gcttaggcta ggaattcaat aaaagatctt tattttcatt agatctgtgt gttggttttt
     4381 tgtgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4441 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4501 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4561 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4621 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4681 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4741 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4801 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4861 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4921 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4981 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5041 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     5101 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5161 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5221 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5281 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5341 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5401 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5461 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5521 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5581 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5641 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5701 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5761 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5821 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5881 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5941 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6001 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6061 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6121 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6181 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6241 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6301 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6361 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6421 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6481 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6541 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6601 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6661 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6721 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6781 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6841 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6901 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6961 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7021 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7081 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gct
//
