LOCUS       sequence-224903-        5279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector; encodes GFP as well as a U6-driven gRNA scaffold 
            (SpyCas9).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5279)
  AUTHORS   Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner S, Bietz A, 
            Waldhauer MC, Borner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, 
            Correia BE, Eils R, Niopek D
  TITLE     Engineered anti-CRISPR proteins for optogenetic control of 
            CRISPR-Cas9.
  JOURNAL   Nat Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 
            10.1038/s41592-018-0178-9.
  PUBMED    30377362
REFERENCE   2  (bases 1 to 5279)
  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: "Nat 
            Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 
            10.1038/s41592-018-0178-9."
COMMENT     Sequence Label: pAAV-RSV-GFP-U6-gRNA scaffold (SpyCas9)
FEATURES             Location/Qualifiers
     source          1..5279
                     /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        2626..2874
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2626..2646
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2797..2816
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(join(3059..3772,3773..3775,3776..3778))
                     /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(3062..3772,3773..3775,3776..3778))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3104..3125)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3453..3472
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3712..3733
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3775..3784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3822..4083)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     4360..4379
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   4452..4581
                     /label=AAV2 ITR
     primer_bind     complement(4637..4656)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4638..4656)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4666..4683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4666..4682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4675..4697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4824..5252
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4911..4930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5121..5142
                     /label=F1ori-F
                     /note="F1 origin, forward 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 ctccgccctg gagaccwtwg gtctccaggg ccgagtgagt gagcgagcgc
     2341 gcatagaggg agtgggtacc tccatcatct ggtttcccag atctgatatc ggcgcgcccg
     2401 agtccaacac ccgtgggaat cccatgggca ccatggcccc tcgctccaaa aatgctttcg
     2461 cgtcgcgcag acactgctcg gtagtttcgg ggatcagcgt ttgagtaaga gcccgcgtct
     2521 gaaccctccg cgccgccccg gccccagtgg aaagacgcgc aggcaaaacg caccacgtga
     2581 cggagcgtga ccgcgcgccg agcgcgcgcc aaggtcgggc aggaagaggg cctatttccc
     2641 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     2701 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     2761 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     2821 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgagtct
     2881 tcgaggaaga caggtttaag agctatgctg gaaacagcat agcaagttta aataaggcta
     2941 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgcgg ccgcctcgag
     3001 tgatcaaaaa accaacacac gcttccaatg aaaataaacg atcctttatt gctagccttt
     3061 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     3121 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     3181 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     3241 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     3301 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     3361 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     3421 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     3481 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     3541 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     3601 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     3661 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     3721 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg
     3781 tggcgaccgg tggatcccgg gcccgcggta cccagcttgg aggtgcacac caatgtggtg
     3841 aatggtcaaa tggcgtttat tgtatcgagc taggcactta aatacaatat ctctgcaatg
     3901 cggaattcag tggttcgtcc aatccatgtc agacccgtct gttgccttcc taataaggca
     3961 cgatcgtacc accttacttc caccaatcgg catgcacggt gctttttctc tccttgtaag
     4021 gcatgttgct aactcatcgt taccatgttg caagactaca agagtattgc ataagactac
     4081 atttccccct ccctatgcaa aagcgaaact actatatcct gaggggactc ctaaccgcgt
     4141 acaaccgaag ccccgctttt cgcctaaaca caccctagtc ccctcagata cgcgtatatc
     4201 tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc ttcatgcaat
     4261 tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac tcagcgacct
     4321 ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag cagattgtac
     4381 tgagagtgca ccatacggat ctgcgatgat aagctgtcaa acatgagaat tggtcgacga
     4441 tatcagatct gaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4501 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4561 agtgagcgag cgagcgcgca gagagggagt ggcccccccc cccccccccc cntgcattct
     4621 agagagctcc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     4681 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4741 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4801 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4861 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4921 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4981 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5041 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5101 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5161 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5221 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattt
//
