LOCUS       sequence-224913-        5280 bp    DNA     circular SYN 15-APR-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 5280)
  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 5280)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..5280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     624..643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        624..642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   699..828
                     /label=AAV2 ITR
     primer_bind     complement(901..920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1197..1458
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     regulatory      1496..1505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1502..1504,1505..1507,1508..2221)
                     /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(1502..1504,1505..1507,1508..2218)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1547..1568)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1808..1827)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2155..2176
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2407..2655)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2465..2484)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2635..2655)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3092..3112)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3092..3110)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3131..3147)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3131..3147)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3144..3166)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3155..3171
                     /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(3179..3185,3186..3203,3204..3209))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3224..3245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3533..4121)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3613..3632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4292..5083,5084..5152))
                     /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     4915..4934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5153..5257)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcacwttc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg gagctctcta
      661 gaatgcaggg gggggggggg ggggkgncca ctccctctct gcgcgctcgc tcgctcactg
      721 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
      781 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctca gatctgatat
      841 cgtcgaccaa ttctcatgtt tgacagctta tcatcgcaga tccgtatggt gcactctcag
      901 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      961 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     1021 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     1081 atatacgcgt atctgagggg actagggtgt gtttaggcga aaagcggggc ttcggttgta
     1141 cgcggttagg agtcccctca ggatatagta gtttcgcttt tgcataggga gggggaaatg
     1201 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     1261 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     1321 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattccgc
     1381 attgcagaga tattgtattt aagtgcctag ctcgatacaa taaacgccat ttgaccattc
     1441 accacattgg tgtgcacctc caagctgggt accgcgggcc cgggatccac cggtcgccac
     1501 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1561 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1621 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1681 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1741 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1801 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1861 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1921 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1981 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2041 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2101 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2161 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     2221 aaggctagca ataaaggatc gtttattttc attggaagcg tgtgttggtt ttttgatcac
     2281 tcgaggcggc cgcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact
     2341 agccttattt aaacttgcta tgctgtttcc agcatagctc ttaaacggat tatgcctggc
     2401 accattggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca
     2461 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc
     2521 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa
     2581 attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat
     2641 gggaaatagg ccctcttcct gcccgacctt ggcgcgcgct cggcgcgcgg tcacgctccg
     2701 tcacgtggtg cgttttgcct gcgcgtcttt ccactggggc cggggcggcg cggagggttc
     2761 agacgcgggc tcttactcaa acgctgatcc ccgaaactac cgagcagtgt ctgcgcgacg
     2821 cgaaagcatt tttggagcga ggggccatgg tgcccatggg attcccacgg gtgttggact
     2881 cgggcgcgcc gatatcagat ctgggaaacc agatgatgga ggtacccact ccctctatgc
     2941 gcgctcgctc actcactcgg ccctggagac caaaggtctc cagsctggag accaaaggtc
     3001 tccagggccg agtgagtgag cgagcgcgca tagagggagt gggtaggacg cgtcctgcag
     3061 gatgcatact agtggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     3121 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3181 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3241 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3301 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3361 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3421 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3481 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3541 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3601 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3661 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3721 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3781 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3841 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3901 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3961 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4021 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4081 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4141 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4201 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4261 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4321 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4381 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4441 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4501 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4561 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4621 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4681 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4741 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4801 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4861 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4921 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4981 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5041 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5101 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5161 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5221 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
