LOCUS       sequence-232740-        5261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector; encodes GFP as well as a U6-driven Rosa26-targeting gRNA
            (SpyCas9 scaffold).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5261)
  AUTHORS   Hoffmann MD, Aschenbrenner S, Grosse S, Rapti K, Domenger C, Fakhiri
            J, Mastel M, Borner K, Eils R, Grimm D, Niopek D
  TITLE     Cell-specific CRISPR-Cas9 activation by microRNA-dependent 
            expression of anti-CRISPR proteins.
  JOURNAL   Nucleic Acids Res. 2019 Apr 15. pii: 5458120. doi: 
            10.1093/nar/gkz271.
  PUBMED    30982889
REFERENCE   2  (bases 1 to 5261)
  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: "Nucleic 
            Acids Res. 2019 Apr 15. pii: 5458120. doi: 10.1093/nar/gkz271."
COMMENT     Sequence Label: pAAV-RSV-GFP-U6-Rosa26 gRNA (SpyCas9 scaffold)
FEATURES             Location/Qualifiers
     source          1..5261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2604..2852
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2604..2624
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2775..2794
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(join(3039..3752,3753..3755,3756..3758))
                     /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(3042..3752,3753..3755,3756..3758))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3084..3105)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3433..3452
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3692..3713
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3755..3764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3802..4063)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     4340..4359
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   4432..4561
                     /label=AAV2 ITR
     primer_bind     complement(4617..4636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4618..4636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4646..4663)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4646..4662)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4655..4677)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4803..5258)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4940..4961)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5152..5171
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccact agtatgcatc
     2221 ctgcaggacg cgtcctaccc actccctcta tgcgcgctcg ctcactcact cggccctgga
     2281 gaccaaaggt ctccagggcc gagtgagtga gcgagcgcgc atagagggag tgggtacctc
     2341 catcatctgg tttcccagat ctgatatcgg cgcgcccgag tccaacaccc gtgggaatcc
     2401 catgggcacc atggcccctc gctccaaaaa tgctttcgcg tcgcgcagac actgctcggt
     2461 agtttcgggg atcagcgttt gagtaagagc ccgcgtctga accctccgcg ccgccccggc
     2521 cccagtggaa agacgcgcag gcaaaacgca ccacgtgacg gagcgtgacc gcgcgccgag
     2581 cgcgcgccaa ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata
     2641 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     2701 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     2761 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     2821 ctttatatat cttgtggaaa ggacgaaaca ccgactccag tctttctaga agagtttaag
     2881 agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa
     2941 agtggcaccg agtcggtgct ttttttgcgg ccgcctcgag tgatcaaaaa accaacacac
     3001 gcttccaatg aaaataaacg atcctttatt gctagccttt acttgtacag ctcgtccatg
     3061 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     3121 ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg
     3181 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc
     3241 tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg
     3301 atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc
     3361 tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc
     3421 acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg
     3481 tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg
     3541 ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc
     3601 ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc
     3661 tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc
     3721 accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggcgaccgg tggatcccgg
     3781 gcccgcggta cccagcttgg aggtgcacac caatgtggtg aatggtcaaa tggcgtttat
     3841 tgtatcgagc taggcactta aatacaatat ctctgcaatg cggaattcag tggttcgtcc
     3901 aatccatgtc agacccgtct gttgccttcc taataaggca cgatcgtacc accttacttc
     3961 caccaatcgg catgcacggt gctttttctc tccttgtaag gcatgttgct aactcatcgt
     4021 taccatgttg caagactaca agagtattgc ataagactac atttccccct ccctatgcaa
     4081 aagcgaaact actatatcct gaggggactc ctaaccgcgt acaaccgaag ccccgctttt
     4141 cgcctaaaca caccctagtc ccctcagata cgcgtatatc tggcccgtac atcgcgaagc
     4201 agcgcaaaac gcctaaccct aagcagattc ttcatgcaat tgtcggtcaa gccttgcctt
     4261 gttgtagctt aaattttgct cgcgcactac tcagcgacct ccaacacaca agcagggagc
     4321 agatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatacggat
     4381 ctgcgatgat aagctgtcaa acatgagaat tggtcgacga tatcagatct gaggaacccc
     4441 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4501 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4561 gagagggagt ggcccccccc cccccccccc cctgcattct agagagctcc aattcgccct
     4621 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4681 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     4741 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     4801 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     4861 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4921 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4981 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5041 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5101 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5161 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5221 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca g
//
