LOCUS       sequence-297790-        6783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector expressed CasRx and sgRNA_LACZ for AAV package.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6783)
  AUTHORS   He B, Peng W, Huang J, Zhang H, Zhou Y, Yang X, Liu J, Li Z, Xu C, 
            Xue M, Yang H, Huang P
  TITLE     Modulation of metabolic functions through Cas13d-mediated gene 
            knockdown in liver.
  JOURNAL   Protein Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi: 
            10.1007/s13238-020-00700-2.
  PUBMED    32185621
REFERENCE   2  (bases 1 to 6783)
  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"; journalName: "Protein 
            Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi: 
            10.1007/s13238-020-00700-2."
COMMENT     Sequence Label: AAV-EFS-NLS-CasRx-U6-DR-sgRNA_LacZ-DR
FEATURES             Location/Qualifiers
     source          1..6783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        186..397
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             414..434
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             444..3341
                     /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             3342..3365
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3366..3386
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3387..3611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3639..3887
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3639..3659
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3810..3829
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3889..3924
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        3955..3990
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   4046..4186
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4046..4175
                     /label=AAV2 ITR
     rep_origin      4261..4716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4348..4367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4558..4579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4733..4752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4852..4874
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4912..4930)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4998..5102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5103..5171,5172..5963)
                     /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(5321..5340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6134..6722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6623..6642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gataggtctt gaaaggagtg ggaattggct ccggtgcccg
      181 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      241 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      301 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      361 cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggtgccacc atgcctaaaa
      421 agaaaagaaa ggtgggttct ggtatcgaga agaagaagag cttcgccaag ggcatgggag
      481 tgaagagcac cctggtgtcc ggctctaagg tgtacatgac cacatttgct gagggaagcg
      541 acgccaggct ggagaagatc gtggagggcg atagcatcag atccgtgaac gagggagagg
      601 ctttcagcgc cgagatggct gacaagaacg ctggctacaa gatcggaaac gccaagtttt
      661 cccacccaaa gggctacgcc gtggtggcta acaacccact gtacaccgga ccagtgcagc
      721 aggacatgct gggactgaag gagacactgg agaagaggta cttcggcgag tccgccgacg
      781 gaaacgataa catctgcatc caggtcatcc acaacatcct ggatatcgag aagatcctgg
      841 ctgagtacat cacaaacgcc gcttacgccg tgaacaacat ctccggcctg gacaaggata
      901 tcatcggctt cggaaagttt tctaccgtgt acacatacga cgagttcaag gatccagagc
      961 accaccgggc cgcttttaac aacaacgaca agctgatcaa cgccatcaag gctcagtacg
     1021 acgagttcga taactttctg gataacccca ggctgggcta cttcggacag gctttctttt
     1081 ctaaggaggg cagaaactac atcatcaact acggaaacga gtgttacgac atcctggccc
     1141 tgctgagcgg actgaggcac tgggtggtgc acaacaacga ggaggagtct cggatcagcc
     1201 gcacctggct gtacaacctg gacaagaacc tggataacga gtacatctcc acactgaact
     1261 acctgtacga caggatcacc aacgagctga caaacagctt ctccaagaac tctgccgcta
     1321 acgtgaacta catcgctgag accctgggca tcaacccagc tgagttcgct gagcagtact
     1381 tcagattttc catcatgaag gagcagaaga acctgggctt caacatcaca aagctgagag
     1441 aagtgatgct ggacagaaag gatatgtccg agatcaggaa gaaccacaag gtgttcgatt
     1501 ctatcagaac caaggtgtac acaatgatgg actttgtgat ctacaggtac tacatcgagg
     1561 aggatgccaa ggtggccgct gccaacaaga gcctgcccga caacgagaag tctctgagcg
     1621 agaaggatat cttcgtgatc aacctgagag gctcctttaa cgacgatcag aaggacgctc
     1681 tgtactacga tgaggccaac aggatctgga gaaagctgga gaacatcatg cacaacatca
     1741 aggagttccg gggaaacaag acccgcgagt acaagaagaa ggacgctcca aggctgccta
     1801 ggatcctgcc tgctggaagg gacgtgagcg ccttcagcaa gctgatgtac gccctgacaa
     1861 tgtttctgga cggaaaggag atcaacgatc tgctgaccac actgatcaac aagttcgaca
     1921 acatccagtc ttttctgaaa gtgatgcctc tgatcggcgt gaacgctaag ttcgtggagg
     1981 agtacgcctt ctttaaggac agcgccaaga tcgctgatga gctgcggctg atcaagtcct
     2041 ttgccaggat gggagagcca atcgctgacg ctaggagagc tatgtacatc gatgccatcc
     2101 ggatcctggg aaccaacctg tcttacgacg agctgaaggc tctggccgac accttcagcc
     2161 tggatgagaa cggcaacaag ctgaagaagg gcaagcacgg aatgcgcaac ttcatcatca
     2221 acaacgtgat cagcaacaag cggtttcact acctgatcag atacggcgac ccagctcacc
     2281 tgcacgagat cgctaagaac gaggccgtgg tgaagttcgt gctgggacgg atcgccgata
     2341 tccagaagaa gcagggccag aacggaaaga accagatcga ccgctactac gagacctgca
     2401 tcggcaagga taagggaaag tccgtgtctg agaaggtgga cgctctgacc aagatcatca
     2461 caggcatgaa ctacgaccag ttcgataaga agagatctgt gatcgaggac accggaaggg
     2521 agaacgccga gagagagaag tttaagaaga tcatcagcct gtacctgaca gtgatctacc
     2581 acatcctgaa gaacatcgtg aacatcaacg ctagatacgt gatcggcttc cactgcgtgg
     2641 agcgcgatgc ccagctgtac aaggagaagg gatacgacat caacctgaag aagctggagg
     2701 agaagggctt tagctccgtg accaagctgt gcgctggaat cgacgagaca gcccccgaca
     2761 agaggaagga tgtggagaag gagatggccg agagagctaa ggagagcatc gactccctgg
     2821 agtctgctaa ccctaagctg tacgccaact acatcaagta ctccgatgag aagaaggccg
     2881 aggagttcac caggcagatc aacagagaga aggccaagac cgctctgaac gcctacctga
     2941 ggaacacaaa gtggaacgtg atcatccggg aggacctgct gcgcatcgat aacaagacct
     3001 gtacactgtt ccggaacaag gctgtgcacc tggaggtggc tcgctacgtg cacgcctaca
     3061 tcaacgacat cgccgaggtg aactcctact ttcagctgta ccactacatc atgcagagga
     3121 tcatcatgaa cgagagatac gagaagtcta gcggcaaggt gtctgagtac ttcgacgccg
     3181 tgaacgatga gaagaagtac aacgatagac tgctgaagct gctgtgcgtg cctttcggat
     3241 actgtatccc acggtttaag aacctgagca tcgaggccct gttcgaccgc aacgaggctg
     3301 ccaagtttga taaggagaag aagaaggtga gcggcaactc cgattacaaa gacgatgacg
     3361 ataagcccaa gaagaagagg aaagtcctgt gccttctagt tgccagccat ctgttgtttg
     3421 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3481 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3541 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3601 gggctctatg ggatgggctg gccttttgct cagctagcga gggcctattt cccatgattc
     3661 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     3721 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3781 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3841 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca agtaaacccc
     3901 taccaactgg tcggggtttg aaaccgtctg gccttcctgt agccagcttt catccaagta
     3961 aacccctacc aactggtcgg ggtttgaaac tttttttgaa ttctgatgcg gtattttctc
     4021 cttacgctga tgcggtggcg gccgcaggaa cccctagtga tggagttggc cactccctct
     4081 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4141 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     4201 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     4261 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4321 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4381 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4441 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4501 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4561 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     4621 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     4681 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     4741 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     4801 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4861 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4921 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5341 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5641 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     5821 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6241 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6781 tgt
//
