LOCUS       sequence-222770-        6755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-CRISPR knockout plasmid for direct in vivo genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6755)
  AUTHORS   Wang G, Chow RD, Ye L, Guzman CD, Dai X, Dong MB, Zhang F, Sharp PA,
            Platt RJ, Chen S
  TITLE     Mapping a functional cancer genome atlas of tumor suppressors in 
            mouse liver using AAV-CRISPR-mediated direct in vivo screening.
  JOURNAL   Sci Adv. 2018 Feb 28;4(2):eaao5508. doi: 10.1126/sciadv.aao5508. 
            eCollection 2018 Feb.
  PUBMED    29503867
REFERENCE   2  (bases 1 to 6755)
  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: "Sci Adv."; 
            date: "2018-02-28"; volume: "4(2):eaao5508. doi"; pages: " 
            10.1126/sciadv.aao5508. eCollection 2018 Feb"
COMMENT     Sequence Label: EFS-Vector
FEATURES             Location/Qualifiers
     source          1..6755
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        503..751
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        770..845
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        900..1111
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1119..1128
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1125..2774
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1191..1209)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             2790..2846
                     /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             2847..2867
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3894..3920
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3942..3990
                     /note="synthetic polyadenylation signal"
     repeat_region   4018..4158
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4018..4147
                     /label=AAV2 ITR
     rep_origin      4233..4688
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4320..4339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4530..4551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4705..4724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4824..4846
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4884..4902)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4970..5074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5075..5143,5144..5935)
                     /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(5293..5312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6106..6694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6595..6614
                     /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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtgtaat agctcctgca tgggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag aagagggcct atttcccatg attccttcat atttgcatat
      541 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      721 tttatatatc ttgtggaaag gacgaaacac cggaagagcg agctcttctg ttttagagct
      781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      841 ggtgcttttt tggtaccagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      901 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      961 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     1021 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     1081 ttttcgcaac gggtttgccg ccagaacaca ggcgtacggc caccatggaa gacgccaaaa
     1141 acataaagaa aggcccggcg ccattctatc cgctggaaga tggaaccgct ggagagcaac
     1201 tgcataaggc tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac
     1261 atatcgaggt ggacatcact tacgctgagt acttcgaaat gtccgttcgg ttggcagaag
     1321 ctatgaaacg atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc
     1381 ttcaattctt tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga
     1441 acgacattta taatgaacgt gaattgctca acagtatggg catttcgcag cctaccgtgg
     1501 tgttcgtttc caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaag ctcccaatca
     1561 tccaaaaaat tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca
     1621 cgttcgtcac atctcatcta cctcccggtt ttaatgaata cgattttgtg ccagagtcct
     1681 tcgataggga caagacaatt gcactgatca tgaactcctc tggatctact ggtctgccta
     1741 aaggtgtcgc tctgcctcat agaactgcct gcgtgagatt ctcgcatgcc agagatccta
     1801 tttttggcaa tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg
     1861 gttttggaat gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt
     1921 atagatttga agaggagctg tttctgagga gccttcagga ttacaagatt caaagtgcgc
     1981 tgctggtgcc aaccctattc tccttcttcg ccaaaagcac tctgattgac aaatacgatt
     2041 tatctaattt acacgaaatt gcttctggtg gcgctcccct ctctaaggaa gtcggggaag
     2101 cggttgccaa gaggttccat ctgccaggta tcaggcaagg atatgggctc actgagacta
     2161 catcagctat tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg
     2221 ttccattttt tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc
     2281 aaagaggcga actgtgtgtg agaggtccta tgattatgtc cggttatgta aacaatccgg
     2341 aagcgaccaa cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact
     2401 gggacgaaga cgaacacttc ttcatcgttg accgcctgaa gtctctgatt aagtacaaag
     2461 gctatcaggt ggctcccgct gaattggaat ccatcttgct ccaacacccc aacatcttcg
     2521 acgcaggtgt cgcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg
     2581 ttttggagca cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag
     2641 taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc
     2701 ttaccggaaa actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg
     2761 gaaagatcgc cgtggctagc ggaagcggag ccactaactt ctccctgttg aaacaagcag
     2821 gggatgtcga agagaatccc gggccaccca agaagaagag gaaggtgtcc aatctcctga
     2881 ctgttcacca gaacctccct gcgctgccag tagatgccac tagcgatgag gtcaggaaaa
     2941 atctcatgga tatgtttagg gatagacagg cgttttctga acacacctgg aaaatgctgc
     3001 ttagcgtgtg ccgatcctgg gcagcctggt gtaagctgaa caatcgcaaa tggttccccg
     3061 ccgagccgga ggacgtgcgc gattacctgc tgtatctcca ggcaagaggg ctggctgtca
     3121 agactatcca gcagcacttg ggccaactga atatgctgca tcgacgcagc gggctccccc
     3181 ggcctagcga ttcaaacgca gtctcccttg ttatgaggag aattagaaag gaaaacgtag
     3241 atgcgggtga gagggctaag caggctctcg cttttgagcg gactgatttc gaccaggtca
     3301 gatccctgat ggagaacagc gatcggtgcc aggacatcag gaacctcgca tttctgggaa
     3361 ttgcatataa cacacttctg cgcatagctg agatcgcccg gatcagagtg aaagacatca
     3421 gtcgaacgga cggcggccgg atgcttattc atattggacg cacaaagaca ttggtcagca
     3481 ccgctggcgt tgaaaaggcc ttgtccctgg gcgtaacgaa gctggtggaa agatggatct
     3541 cagtgtccgg cgtggctgac gaccctaata attacttgtt ctgtcgagtg agaaaaaacg
     3601 gagtcgccgc gccctctgcc accagccaat tgagtacacg ggcccttgaa gggatctttg
     3661 aggcaaccca ccgactcata tacggagcca aggatgacag tggccagagg tatctcgcct
     3721 ggtcaggtca ttctgctagg gtgggggccg cacgagacat ggcgcgggca ggagtctcca
     3781 taccagagat tatgcaagct ggaggttgga caaatgtgaa catcgttatg aactatatcc
     3841 gcaatcttga ctctgaaacc ggggccatgg tgagactgct cgaagatggt gactacccat
     3901 acgatgttcc agattacgct taagaattcg atatcaagct taataaaaga tctttatttt
     3961 cattagatct gtgtgttggt tttttgtgtg gtaaccacgt gcggaccgag cggccgcagg
     4021 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4081 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4141 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4201 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4261 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4321 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4381 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4441 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4501 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4561 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     4621 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     4681 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     4741 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4801 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4861 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     4921 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4981 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5041 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5101 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5161 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5221 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5281 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5341 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5401 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5461 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5521 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5581 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5641 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5701 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5761 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5821 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5881 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5941 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6001 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6061 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6121 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6181 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6241 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6301 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6361 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6421 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6481 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6541 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6601 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6661 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6721 gttcctggcc ttttgctggc cttttgctca catgt
//
