LOCUS       sequence-206304-        6766 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 6766)
  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 6766)
  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..6766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        514..762
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        781..856
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        911..1122
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1130..1139
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1136..2785
                     /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(1202..1220)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             2801..2857
                     /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             2858..2878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3905..3931
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3953..4001
                     /note="synthetic polyadenylation signal"
     repeat_region   4029..4169
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4029..4158
                     /label=AAV2 ITR
     rep_origin      4244..4699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4541..4562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4716..4735)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4835..4857
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4895..4913)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4981..5085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5086..5154,5155..5946)
                     /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(5304..5323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6117..6705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6606..6625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgtaa tagctcctgc
      421 atgggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      481 aagtggcacc gagtcggtgc tttttttcta gaagagggcc tatttcccat gattccttca
      541 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      601 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      661 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      721 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggaagagc gagctcttct
      781 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      841 ggcaccgagt cggtgctttt ttggtaccag gtcttgaaag gagtgggaat tggctccggt
      901 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      961 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     1021 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     1081 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggcgtacgg ccaccatgga
     1141 agacgccaaa aacataaaga aaggcccggc gccattctat ccgctggaag atggaaccgc
     1201 tggagagcaa ctgcataagg ctatgaagag atacgccctg gttcctggaa caattgcttt
     1261 tacagatgca catatcgagg tggacatcac ttacgctgag tacttcgaaa tgtccgttcg
     1321 gttggcagaa gctatgaaac gatatgggct gaatacaaat cacagaatcg tcgtatgcag
     1381 tgaaaactct cttcaattct ttatgccggt gttgggcgcg ttatttatcg gagttgcagt
     1441 tgcgcccgcg aacgacattt ataatgaacg tgaattgctc aacagtatgg gcatttcgca
     1501 gcctaccgtg gtgttcgttt ccaaaaaggg gttgcaaaaa attttgaacg tgcaaaaaaa
     1561 gctcccaatc atccaaaaaa ttattatcat ggattctaaa acggattacc agggatttca
     1621 gtcgatgtac acgttcgtca catctcatct acctcccggt tttaatgaat acgattttgt
     1681 gccagagtcc ttcgataggg acaagacaat tgcactgatc atgaactcct ctggatctac
     1741 tggtctgcct aaaggtgtcg ctctgcctca tagaactgcc tgcgtgagat tctcgcatgc
     1801 cagagatcct atttttggca atcaaatcat tccggatact gcgattttaa gtgttgttcc
     1861 attccatcac ggttttggaa tgtttactac actcggatat ttgatatgtg gatttcgagt
     1921 cgtcttaatg tatagatttg aagaggagct gtttctgagg agccttcagg attacaagat
     1981 tcaaagtgcg ctgctggtgc caaccctatt ctccttcttc gccaaaagca ctctgattga
     2041 caaatacgat ttatctaatt tacacgaaat tgcttctggt ggcgctcccc tctctaagga
     2101 agtcggggaa gcggttgcca agaggttcca tctgccaggt atcaggcaag gatatgggct
     2161 cactgagact acatcagcta ttctgattac acccgagggg gatgataaac cgggcgcggt
     2221 cggtaaagtt gttccatttt ttgaagcgaa ggttgtggat ctggataccg ggaaaacgct
     2281 gggcgttaat caaagaggcg aactgtgtgt gagaggtcct atgattatgt ccggttatgt
     2341 aaacaatccg gaagcgacca acgccttgat tgacaaggat ggatggctac attctggaga
     2401 catagcttac tgggacgaag acgaacactt cttcatcgtt gaccgcctga agtctctgat
     2461 taagtacaaa ggctatcagg tggctcccgc tgaattggaa tccatcttgc tccaacaccc
     2521 caacatcttc gacgcaggtg tcgcaggtct tcccgacgat gacgccggtg aacttcccgc
     2581 cgccgttgtt gttttggagc acggaaagac gatgacggaa aaagagatcg tggattacgt
     2641 cgccagtcaa gtaacaaccg cgaaaaagtt gcgcggagga gttgtgtttg tggacgaagt
     2701 accgaaaggt cttaccggaa aactcgacgc aagaaaaatc agagagatcc tcataaaggc
     2761 caagaagggc ggaaagatcg ccgtggctag cggaagcgga gccactaact tctccctgtt
     2821 gaaacaagca ggggatgtcg aagagaatcc cgggccaccc aagaagaaga ggaaggtgtc
     2881 caatctcctg actgttcacc agaacctccc tgcgctgcca gtagatgcca ctagcgatga
     2941 ggtcaggaaa aatctcatgg atatgtttag ggatagacag gcgttttctg aacacacctg
     3001 gaaaatgctg cttagcgtgt gccgatcctg ggcagcctgg tgtaagctga acaatcgcaa
     3061 atggttcccc gccgagccgg aggacgtgcg cgattacctg ctgtatctcc aggcaagagg
     3121 gctggctgtc aagactatcc agcagcactt gggccaactg aatatgctgc atcgacgcag
     3181 cgggctcccc cggcctagcg attcaaacgc agtctccctt gttatgagga gaattagaaa
     3241 ggaaaacgta gatgcgggtg agagggctaa gcaggctctc gcttttgagc ggactgattt
     3301 cgaccaggtc agatccctga tggagaacag cgatcggtgc caggacatca ggaacctcgc
     3361 atttctggga attgcatata acacacttct gcgcatagct gagatcgccc ggatcagagt
     3421 gaaagacatc agtcgaacgg acggcggccg gatgcttatt catattggac gcacaaagac
     3481 attggtcagc accgctggcg ttgaaaaggc cttgtccctg ggcgtaacga agctggtgga
     3541 aagatggatc tcagtgtccg gcgtggctga cgaccctaat aattacttgt tctgtcgagt
     3601 gagaaaaaac ggagtcgccg cgccctctgc caccagccaa ttgagtacac gggcccttga
     3661 agggatcttt gaggcaaccc accgactcat atacggagcc aaggatgaca gtggccagag
     3721 gtatctcgcc tggtcaggtc attctgctag ggtgggggcc gcacgagaca tggcgcgggc
     3781 aggagtctcc ataccagaga ttatgcaagc tggaggttgg acaaatgtga acatcgttat
     3841 gaactatatc cgcaatcttg actctgaaac cggggccatg gtgagactgc tcgaagatgg
     3901 tgactaccca tacgatgttc cagattacgc ttaagaattc gatatcaagc ttaataaaag
     3961 atctttattt tcattagatc tgtgtgttgg ttttttgtgt ggtaaccacg tgcggaccga
     4021 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4081 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4141 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4201 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4261 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4321 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4381 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4441 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4501 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4561 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4621 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4681 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4741 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4801 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4861 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4921 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     6301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
