LOCUS       sequence-232734-        7600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV Vector for expression of N-terminal SpyCas9 fragment with Intein
            and a U6-driven F+E gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7600)
  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 7600)
  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: AAV N-SpyCas9-Intein, U6-gRNA  scaffold (F+E)
FEATURES             Location/Qualifiers
     source          1..7600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          676..808
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      838..847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             850..870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             895..2610
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(3350..3369)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3379..3460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3404..3423
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3809..4057
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3809..3829
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3980..3999
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4224..4353
                     /label=AAV2 ITR
     primer_bind     complement(4391..4407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4391..4407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4404..4426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4415..4431
                     /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(4439..4445,4446..4463,4464..4469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4484..4505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4793..5381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4873..4892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5552..6343,6344..6412))
                     /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     6175..6194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6413..6517)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6543..6998)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6680..6701)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6892..6911
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7125..7147
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7139..7156
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7140..7156
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7175..7304
                     /label=AAV2 ITR
     primer_bind     complement(7351..7371)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc
      601 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      661 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa
      721 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact
      781 gacatccact ttgcctttct ctccacaggg gtaccgaagc cgctagcgct accggtcgcc
      841 accatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag
      901 aagtactcca ttgggctcga tatcggcaca aacagcgtcg gctgggccgt cattacggac
      961 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata
     1021 aagaagaacc tcattggcgc cctcctgttc gactccgggg aaacggccga agccacgcgg
     1081 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag
     1141 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag
     1201 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg
     1261 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta
     1321 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa
     1381 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa
     1441 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca
     1501 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa
     1561 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc
     1621 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag
     1681 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc
     1741 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt
     1801 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag
     1861 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg
     1921 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt
     1981 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg
     2041 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc
     2101 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc
     2161 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc
     2221 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg
     2281 tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat
     2341 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct
     2401 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag
     2461 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag
     2521 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag
     2581 ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg
     2641 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac
     2701 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt
     2761 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc
     2821 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca
     2881 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt
     2941 aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc
     3001 tttaaggagg acatccagaa agcacaagtt tgtctggctg gcgatactct cattaccctg
     3061 gccgatggac gacgagtgcc tattagagaa ctggtgtcac agcagaattt ttccgtgtgg
     3121 gctctgaatc ctcagactta ccgcctggag agggctagag tgagtagagc tttctgtacc
     3181 ggcatcaaac ctgtgtaccg cctcaccact agactgggga gatccattag ggccactgcc
     3241 aaccaccgat ttctcacacc tcagggctgg aaacgagtcg atgaactcca gcctggagat
     3301 tacctggctc tgcctaggag aatccctact gcctcctgat ctcgagctcg atgagtttgg
     3361 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     3421 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     3481 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     3541 caaatgtggt acttaagagg gggagaccaa agggcacgcg tcgagtccaa cacccgtggg
     3601 aatcccatgg gcaccatggc ccctcgctcc aaaaatgctt tcgcgtcgcg cagacactgc
     3661 tcggtagttt cggggatcag cgtttgagta agagcccgcg tctgaaccct ccgcgccgcc
     3721 ccggccccag tggaaagacg cgcaggcaaa acgcaccacg tgacggagcg tgaccgcgcg
     3781 ccgagcgcgc gccaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt
     3841 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     3901 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     3961 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     4021 cttggcttta tatatcttgt ggaaaggacg aaacaccgag tcttcgagga agacaggttt
     4081 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     4141 aaaagtggca ccgagtcggt gctttttttc tagagggcgg ccgcaggtag ataagtagca
     4201 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
     4261 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4321 ccgggcggcc tcagtgagcg agcgagcgcg cagcctaatt aaggccttaa ttaaatctgg
     4381 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4441 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4501 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4561 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4621 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4681 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4741 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4801 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4861 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4921 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4981 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5041 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5101 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5161 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5221 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5281 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5341 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5401 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5461 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5521 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5581 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5641 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5701 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5761 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5821 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5881 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5941 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6001 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6061 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6121 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6181 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6241 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6301 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6361 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6421 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6481 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6541 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     6601 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     6661 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     6721 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     6781 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     6841 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     6901 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     6961 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     7021 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     7081 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     7141 taaaacgacg gccagtgaat taggttaatt aaggctgcgc gctcgctcgc tcactgaggc
     7201 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     7261 agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt taatgattaa
     7321 cccgccatgc tacttatcta ctgcggccgc ggaatgtgtg tcagttaggg tgtggaaagt
     7381 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     7441 gtcccggtct cctcccatgc atgtcaatat tggccattag ccatattatt cattggttat
     7501 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg
     7561 tacatttata ttggctcatg tccaatatga ccgccatgtt
//
