LOCUS       sequence-349297-        6098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA and can be packaged into AAV particles for somatic 
            delivery into Cas9 transgenic mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6098)
  AUTHORS   Kaltenbacher T, Loprich J, Maresch R, Weber J, Muller S, Oellinger 
            R, Gross N, Griger J, de Andrade Kratzig N, Avramopoulos P, 
            Ramanujam D, Brummer S, Widholz SA, Barthel S, Falcomata C, Pfaus A,
            Alnatsha A, Mayerle J, Schmidt-Supprian M, Reichert M, Schneider G, 
            Ehmer U, Braun CJ, Saur D, Engelhardt S, Rad R
  TITLE     CRISPR somatic genome engineering and cancer modeling in the mouse 
            pancreas and liver.
  JOURNAL   Nat Protoc. 2022 Apr;17(4):1142-1188. doi: 
            10.1038/s41596-021-00677-0. Epub 2022 Mar 14.
  PUBMED    35288718
REFERENCE   2  (bases 1 to 6098)
  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"; doi: 
            "10.1038/s41596-021-00677-0"; journalName: "Nat Protoc"; date: 
            "2022-04"; volume: "17"; issue: "4"; pages: "1142-1188"
COMMENT     Sequence Label: scAAV-sgRNA-GFP
FEATURES             Location/Qualifiers
     source          1..6098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        536..784
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     536..556
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     707..726
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        803..878
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(953..970)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    959..1166
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(1183..1896,1897..1899,1900..1902))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1186..1896,1897..1899,1900..1902))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1228..1249)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1577..1596
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1836..1857
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1899..1908
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1946..2207)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     repeat_region   2635..2764
                     /label=AAV2 ITR
     rep_origin      3135..3648
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3222..3241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3432..3453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4249..4271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4309..4327)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4395..4499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4500..4568,4569..5360)
                     /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(4718..4737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5531..6098,1..21)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6020..6039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
       61 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
      121 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
      181 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
      241 ccgattcatt aatgcagctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg
      301 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
      361 tggtaaaagc ttcagtctcc tgagtagctg ggactacagg agcttgttac cacacccagc
      421 tccagtttat aaattcatct ccagtttata aaggaggaaa ccgaggtact gagaggttaa
      481 aaaaccttcc tgcagacacg cgtcaagctt ctggatccgg taccggctca catgtgaggg
      541 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
      601 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
      661 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
      721 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
      781 caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa ataaggctag
      841 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa
      901 tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaatgatc agcctcgact
      961 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1021 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1081 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1141 gaagagaata gcaggcatgc tggggagcgg ccttgctagc ctttacttgt acagctcgtc
     1201 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     1261 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     1321 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     1381 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     1441 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     1501 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     1561 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     1621 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     1681 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     1741 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     1801 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     1861 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcga ccggtggatc
     1921 ccgggcccgc ggtacccagc ttggaggtgc acaccaatgt ggtgaatggt caaatggcgt
     1981 ttattgtatc gagctaggca cttaaataca atatctctgc aatgcggaat tcagtggttc
     2041 gtccaatcca tgtcagaccc gtctgttgcc ttcctaataa ggcacgatcg taccacctta
     2101 cttccaccaa tcggcatgca cggtgctttt tctctccttg taaggcatgt tgctaactca
     2161 tcgttaccat gttgcaagac tacaagagta ttgcataaga ctacatttcc ccctccctat
     2221 gcaaaagcga aactactata tcctgagggg actcctaacc gcgtacaacc gaagccccgc
     2281 ttttcgccta aacacaccct agtcccctca gatacgcgta tatctggccc gtacatcgcg
     2341 aagcagcgca aaacgcctaa ccctaagcag attcttcatg caattgtcgg tcaagccttg
     2401 ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg
     2461 gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatac
     2521 ggatctgcga tgataagctg tcaaacatga gaattggtcg tcgacctgca ggcatgcaag
     2581 ctctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     2641 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     2701 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     2761 gcagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2821 tgaatggcga atggcgattc cgttgcaatg gctggcggta atattgttct ggatattacc
     2881 agcaaggccg atagtttgag ttcttctact caggcaagtg atgttattac taatcaaaga
     2941 agtattgcga caacggttaa tttgcgtgat ggacagactc ttttactcgg tggcctcact
     3001 gattataaaa acacttctca ggattctggc gtaccgttcc tgtctaaaat ccctttaatc
     3061 ggcctcctgt ttagctcccg ctctgattct aacgaggaaa gcacgttata cgtgctcgtc
     3121 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3181 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3241 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3301 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3361 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3421 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3481 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3541 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aaatatttgc
     3601 ttatacaatc ttcctgtttt tggggctttt ctgattatca accggggtac atatgattga
     3661 catgctagtt ttacgattac cgttcatcga ttctcttgtt tgctccagac tctcaggcaa
     3721 tgacctgata gcctttgtag agacctctca aaaatagcta ccctctccgg catgaattta
     3781 tcagctagaa cggttgaata tcatattgat ggtgatttga ctgtctccgg cctttctcac
     3841 ccgtttgaat ctttacctac acattactca ggcattgcat ttaaaatata tgagggttct
     3901 aaaaattttt atccttgcgt tgaaataaag gcttctcccg caaaagtatt acagggtcat
     3961 aatgtttttg gtacaaccga tttagcttta tgctctgagg ctttattgct taattttgct
     4021 aattctttgc cttgcctgta tgatttattg gatgttggaa tcgcctgatg cggtattttc
     4081 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     4141 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4201 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4261 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4321 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4381 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4441 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4501 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4561 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4621 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4681 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4741 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4801 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4861 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4921 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4981 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5041 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5101 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5161 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5221 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5281 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5341 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5401 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5461 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5521 aaggatcttc ttgagttcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5581 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5641 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5701 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5761 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5821 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5881 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5941 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6001 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6061 acctctgact tgagcgtcga tttttgtgat gctcgtca
//
