LOCUS       sequence-218003-        5285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector; encodes GFP as well as a U6-driven CFTR-targeting gRNA 
            (SpyCas9 scaffold).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5285)
  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 5285)
  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-CFTR gRNA (SpyCas9 scaffold)
FEATURES             Location/Qualifiers
     source          1..5285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(902..921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1198..1461
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     regulatory      1499..1508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1505..1507,1508..1510,1511..2224)
                     /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             join(1505..1507,1508..1510,1511..2221)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1550..1571)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1811..1830)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2158..2179
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2411..2659)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2469..2488)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2639..2659)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3097..3117)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3097..3115)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3136..3152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3136..3152)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3149..3171)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3160..3176
                     /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)."
     promoter        complement(join(3184..3190,3191..3208,3209..3214))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3229..3250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3538..4126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3618..3637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4297..5088,5089..5157))
                     /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     4920..4939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5158..5262)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctctc
      661 tagaatgcag gggggggggg gggggggggc cactccctct ctgcgcgctc gctcgctcac
      721 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
      781 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct agatctgata
      841 tcgtcgacca attctcatgt ttgacagctt atcatcgcag atccgtatgg tgcactctca
      901 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      961 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     1021 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     1081 gatatacgcg tatctgaggg gactagggtg tgtttaggcg aaaagcgggg cttcggttgt
     1141 acgcggttag gagtcccctc aggatatagt agtttcgctt ttgcataggg agggggaaat
     1201 gtagtcttat gcaaatactc ttggtagtct tgcaacatgg taaggatgag ttagcaacat
     1261 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
     1321 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattc
     1381 cgcattgcag agatattgta tttaagtgcc tagctcgata caataaacgc catttgacca
     1441 ttcaccacat tggtgtgcac ctccaagctg ggtaccgcgg gcccgggatc caccggtcgc
     1501 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1561 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1621 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1681 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1741 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1801 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1861 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1921 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1981 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2041 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgttgc tgcccgacaa
     2101 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2161 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2221 gtaaaggcta gcaataaagg atcgtttatt ttcattggaa gcgtgtgttg gttttttgat
     2281 cactcgaggc ggccgcaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg
     2341 actagcctta tttaaacttg ctatgctgtt tccagcatag ctcttaaacg gattatgcct
     2401 ggcaccattc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact
     2461 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact
     2521 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag
     2581 tcaaattaat tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa
     2641 tcatgggaaa taggccctct tcctgcccga ccttggcgcg cgctcggcgc gcggtcacgc
     2701 tccgtcacgt ggtgcgtttt gcctgcgcgt ctttccactg gggccggggc ggcgcggagg
     2761 gttcagacgc gggctcttac tcaaacgctg atccccgaaa ctaccgagca gtgtctgcgc
     2821 gacgcgaaag catttttgga gcgaggggcc atggtgccca tgggattccc acgggtgttg
     2881 gactcgggcg cgccgatatc agatctggca aacctagatg atggaggtac ccactccctc
     2941 tatgcgcgct cgctcactca ctcggccctg ccggccagag gccggcagtc tggagacctt
     3001 tggtctccag ggccgagtga gtgagcgagc gcgcatagag ggagtgggta ggacgcgtcc
     3061 tgcaggatgc atactagtgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     3121 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3181 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3241 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3301 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3361 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3421 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3481 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3541 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3601 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3661 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3721 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3781 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3841 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3901 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3961 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4021 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4081 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4141 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4201 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4261 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4321 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4381 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4441 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4501 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4561 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4621 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4681 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4741 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4801 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4861 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4921 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4981 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5041 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5101 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5161 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5221 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5281 gccac
//
