LOCUS       sequence-206305-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-CRISPR knockout plasmid with Cre recombinase driven by TBG 
            promoter for direct in vivo genome editing in mouse liver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  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 7019)
  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: TBG-Vector
FEATURES             Location/Qualifiers
     source          1..7019
                     /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        925..1334
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      1353..1362
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1383..1392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1389..3038
                     /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(1455..1473)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             3054..3110
                     /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             3111..3131
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4158..4184
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4206..4254
                     /note="synthetic polyadenylation signal"
     repeat_region   4282..4422
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4282..4411
                     /label=AAV2 ITR
     rep_origin      4497..4952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4584..4603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4794..4815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4969..4988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5088..5110
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5148..5166)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5234..5338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5339..5407,5408..6199)
                     /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(5557..5576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6370..6958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6859..6878
                     /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 ttggtaccgc ggcctctaga ctcgaggggc tggaagctac
      901 ctttgacatc atttcctctg cgaatgcatg tataatttct acagaaccta ttagaaagga
      961 tcacccagcc tctgcttttg tacaactttc ccttaaaaaa ctgccaattc cactgctgtt
     1021 tggcccaata gtgagaactt tttcctgctg cctcttggtg cttttgccta tggcccctat
     1081 tctgcctgct gaagacactc ttgccagcat ggacttaaac ccctccagct ctgacaatcc
     1141 tctttctctt ttgttttaca tgaagggtct ggcagccaaa gcaatcactc aaagttcaaa
     1201 ccttatcatt ttttgctttg ttcctcttgg ccttggtttt gtacatcagc tttgaaaata
     1261 ccatcccagg gttaatgctg gggttaattt ataactaaga gtgctctagt tttgcaatac
     1321 aggacatgct ataaaaatgg aaagataccg gtgccaccat ggccccaaag gttaaccgta
     1381 cggccaccat ggaagacgcc aaaaacataa agaaaggccc ggcgccattc tatccgctgg
     1441 aagatggaac cgctggagag caactgcata aggctatgaa gagatacgcc ctggttcctg
     1501 gaacaattgc ttttacagat gcacatatcg aggtggacat cacttacgct gagtacttcg
     1561 aaatgtccgt tcggttggca gaagctatga aacgatatgg gctgaataca aatcacagaa
     1621 tcgtcgtatg cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc gcgttattta
     1681 tcggagttgc agttgcgccc gcgaacgaca tttataatga acgtgaattg ctcaacagta
     1741 tgggcatttc gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga
     1801 acgtgcaaaa aaagctccca atcatccaaa aaattattat catggattct aaaacggatt
     1861 accagggatt tcagtcgatg tacacgttcg tcacatctca tctacctccc ggttttaatg
     1921 aatacgattt tgtgccagag tccttcgata gggacaagac aattgcactg atcatgaact
     1981 cctctggatc tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga
     2041 gattctcgca tgccagagat cctatttttg gcaatcaaat cattccggat actgcgattt
     2101 taagtgttgt tccattccat cacggttttg gaatgtttac tacactcgga tatttgatat
     2161 gtggatttcg agtcgtctta atgtatagat ttgaagagga gctgtttctg aggagccttc
     2221 aggattacaa gattcaaagt gcgctgctgg tgccaaccct attctccttc ttcgccaaaa
     2281 gcactctgat tgacaaatac gatttatcta atttacacga aattgcttct ggtggcgctc
     2341 ccctctctaa ggaagtcggg gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc
     2401 aaggatatgg gctcactgag actacatcag ctattctgat tacacccgag ggggatgata
     2461 aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg gatctggata
     2521 ccgggaaaac gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta
     2581 tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt gattgacaag gatggatggc
     2641 tacattctgg agacatagct tactgggacg aagacgaaca cttcttcatc gttgaccgcc
     2701 tgaagtctct gattaagtac aaaggctatc aggtggctcc cgctgaattg gaatccatct
     2761 tgctccaaca ccccaacatc ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg
     2821 gtgaacttcc cgccgccgtt gttgttttgg agcacggaaa gacgatgacg gaaaaagaga
     2881 tcgtggatta cgtcgccagt caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt
     2941 ttgtggacga agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga
     3001 tcctcataaa ggccaagaag ggcggaaaga tcgccgtggc tagcggaagc ggagccacta
     3061 acttctccct gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca cccaagaaga
     3121 agaggaaggt gtccaatctc ctgactgttc accagaacct ccctgcgctg ccagtagatg
     3181 ccactagcga tgaggtcagg aaaaatctca tggatatgtt tagggataga caggcgtttt
     3241 ctgaacacac ctggaaaatg ctgcttagcg tgtgccgatc ctgggcagcc tggtgtaagc
     3301 tgaacaatcg caaatggttc cccgccgagc cggaggacgt gcgcgattac ctgctgtatc
     3361 tccaggcaag agggctggct gtcaagacta tccagcagca cttgggccaa ctgaatatgc
     3421 tgcatcgacg cagcgggctc ccccggccta gcgattcaaa cgcagtctcc cttgttatga
     3481 ggagaattag aaaggaaaac gtagatgcgg gtgagagggc taagcaggct ctcgcttttg
     3541 agcggactga tttcgaccag gtcagatccc tgatggagaa cagcgatcgg tgccaggaca
     3601 tcaggaacct cgcatttctg ggaattgcat ataacacact tctgcgcata gctgagatcg
     3661 cccggatcag agtgaaagac atcagtcgaa cggacggcgg ccggatgctt attcatattg
     3721 gacgcacaaa gacattggtc agcaccgctg gcgttgaaaa ggccttgtcc ctgggcgtaa
     3781 cgaagctggt ggaaagatgg atctcagtgt ccggcgtggc tgacgaccct aataattact
     3841 tgttctgtcg agtgagaaaa aacggagtcg ccgcgccctc tgccaccagc caattgagta
     3901 cacgggccct tgaagggatc tttgaggcaa cccaccgact catatacgga gccaaggatg
     3961 acagtggcca gaggtatctc gcctggtcag gtcattctgc tagggtgggg gccgcacgag
     4021 acatggcgcg ggcaggagtc tccataccag agattatgca agctggaggt tggacaaatg
     4081 tgaacatcgt tatgaactat atccgcaatc ttgactctga aaccggggcc atggtgagac
     4141 tgctcgaaga tggtgactac ccatacgatg ttccagatta cgcttaagaa ttcgatatca
     4201 agcttaataa aagatcttta ttttcattag atctgtgtgt tggttttttg tgtggtaacc
     4261 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4321 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4381 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4441 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4501 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4561 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4621 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4681 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4741 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4801 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     4861 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4921 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4981 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5041 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5101 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5161 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5221 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5281 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5341 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5401 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5461 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5521 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5581 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5641 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5701 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5761 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5821 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5881 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5941 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6001 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6061 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6121 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6181 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6241 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6301 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6361 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6421 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6481 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6541 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6601 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6661 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6721 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6781 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6841 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6901 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6961 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
