LOCUS       sequence-222776-        7008 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 7008)
  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 7008)
  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..7008
                     /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        914..1323
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      1342..1351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1372..1381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1378..3027
                     /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(1444..1462)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             3043..3099
                     /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             3100..3120
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4147..4173
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4195..4243
                     /note="synthetic polyadenylation signal"
     repeat_region   4271..4411
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4271..4400
                     /label=AAV2 ITR
     rep_origin      4486..4941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4573..4592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4783..4804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4958..4977)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5077..5099
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5137..5155)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5223..5327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5328..5396,5397..6188)
                     /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(5546..5565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6359..6947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6848..6867
                     /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 tggtaccgcg gcctctagac tcgaggggct ggaagctacc tttgacatca
      901 tttcctctgc gaatgcatgt ataatttcta cagaacctat tagaaaggat cacccagcct
      961 ctgcttttgt acaactttcc cttaaaaaac tgccaattcc actgctgttt ggcccaatag
     1021 tgagaacttt ttcctgctgc ctcttggtgc ttttgcctat ggcccctatt ctgcctgctg
     1081 aagacactct tgccagcatg gacttaaacc cctccagctc tgacaatcct ctttctcttt
     1141 tgttttacat gaagggtctg gcagccaaag caatcactca aagttcaaac cttatcattt
     1201 tttgctttgt tcctcttggc cttggttttg tacatcagct ttgaaaatac catcccaggg
     1261 ttaatgctgg ggttaattta taactaagag tgctctagtt ttgcaataca ggacatgcta
     1321 taaaaatgga aagataccgg tgccaccatg gccccaaagg ttaaccgtac ggccaccatg
     1381 gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc
     1441 gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct
     1501 tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt
     1561 cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc
     1621 agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca
     1681 gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg
     1741 cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa
     1801 aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt
     1861 cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt
     1921 gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct
     1981 actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat
     2041 gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt
     2101 ccattccatc acggttttgg aatgtttact acactcggat atttgatatg tggatttcga
     2161 gtcgtcttaa tgtatagatt tgaagaggag ctgtttctga ggagccttca ggattacaag
     2221 attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag cactctgatt
     2281 gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc cctctctaag
     2341 gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca aggatatggg
     2401 ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg
     2461 gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg
     2521 ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat gtccggttat
     2581 gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct acattctgga
     2641 gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct gaagtctctg
     2701 attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt gctccaacac
     2761 cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg tgaacttccc
     2821 gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac
     2881 gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa
     2941 gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag
     3001 gccaagaagg gcggaaagat cgccgtggct agcggaagcg gagccactaa cttctccctg
     3061 ttgaaacaag caggggatgt cgaagagaat cccgggccac ccaagaagaa gaggaaggtg
     3121 tccaatctcc tgactgttca ccagaacctc cctgcgctgc cagtagatgc cactagcgat
     3181 gaggtcagga aaaatctcat ggatatgttt agggatagac aggcgttttc tgaacacacc
     3241 tggaaaatgc tgcttagcgt gtgccgatcc tgggcagcct ggtgtaagct gaacaatcgc
     3301 aaatggttcc ccgccgagcc ggaggacgtg cgcgattacc tgctgtatct ccaggcaaga
     3361 gggctggctg tcaagactat ccagcagcac ttgggccaac tgaatatgct gcatcgacgc
     3421 agcgggctcc cccggcctag cgattcaaac gcagtctccc ttgttatgag gagaattaga
     3481 aaggaaaacg tagatgcggg tgagagggct aagcaggctc tcgcttttga gcggactgat
     3541 ttcgaccagg tcagatccct gatggagaac agcgatcggt gccaggacat caggaacctc
     3601 gcatttctgg gaattgcata taacacactt ctgcgcatag ctgagatcgc ccggatcaga
     3661 gtgaaagaca tcagtcgaac ggacggcggc cggatgctta ttcatattgg acgcacaaag
     3721 acattggtca gcaccgctgg cgttgaaaag gccttgtccc tgggcgtaac gaagctggtg
     3781 gaaagatgga tctcagtgtc cggcgtggct gacgacccta ataattactt gttctgtcga
     3841 gtgagaaaaa acggagtcgc cgcgccctct gccaccagcc aattgagtac acgggccctt
     3901 gaagggatct ttgaggcaac ccaccgactc atatacggag ccaaggatga cagtggccag
     3961 aggtatctcg cctggtcagg tcattctgct agggtggggg ccgcacgaga catggcgcgg
     4021 gcaggagtct ccataccaga gattatgcaa gctggaggtt ggacaaatgt gaacatcgtt
     4081 atgaactata tccgcaatct tgactctgaa accggggcca tggtgagact gctcgaagat
     4141 ggtgactacc catacgatgt tccagattac gcttaagaat tcgatatcaa gcttaataaa
     4201 agatctttat tttcattaga tctgtgtgtt ggttttttgt gtggtaacca cgtgcggacc
     4261 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4321 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4381 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4441 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4501 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4561 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4621 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4681 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4741 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4801 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4861 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4921 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4981 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5041 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5101 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5161 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5221 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5281 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5341 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5401 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5461 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5521 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5581 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5641 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5701 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5761 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5821 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5881 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5941 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6001 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6061 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6121 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6181 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6241 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6301 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6361 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6421 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6481 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6541 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6601 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6661 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6721 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6781 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6841 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6901 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6961 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
