LOCUS       sequence-228610-        7591 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 7591)
  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 7591)
  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..7591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..27)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        257..636
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        637..840
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     791..811
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          932..1064
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1094..1103
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1106..1126
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1151..2866
                     /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(3606..3625)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3635..3716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3660..3679
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4065..4313
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4065..4085
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4236..4255
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4480..4609
                     /label=AAV2 ITR
     primer_bind     complement(4636..4652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4636..4652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4649..4671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4660..4676
                     /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(4684..4690,4691..4708,4709..4714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4729..4750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5038..5626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5118..5137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5797..6588,6589..6657))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6420..6439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6658..6762)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6789..7244)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6926..6947)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7138..7157
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7371..7393
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7385..7402
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7386..7402
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7421..7550
                     /label=AAV2 ITR
ORIGIN
        1 ggccgcggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
       61 gtatgcaaag catgcatctc aattagtcag caaccagtcc cggtctcctc ccatgcatgt
      121 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat
      181 tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca
      241 atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc aattacgggg
      301 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      361 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      421 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      481 cacttggcag tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac
      541 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg
      601 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc
      661 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      721 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc
      781 gccccgttga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagaggt
      841 cgtttagtga accgtcagat cactagtagc tttattgcgg tagtttatca cagttaaatt
      901 gctaacgcag tcagtgctcg actgatcaca ggtaagtatc aaggttacaa gacaggttta
      961 aggaggccaa tagaaactgg gcttgtcgag acagagaaga ttcttgcgtt tctgataggc
     1021 acctattggt cttactgaca tccactttgc ctttctctcc acaggggtac cgaagccgct
     1081 agcgctaccg gtcgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt
     1141 cccagcagcc gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg
     1201 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac
     1261 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggaaac
     1321 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg
     1381 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt
     1441 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat
     1501 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct
     1561 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct
     1621 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa
     1681 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga
     1741 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa
     1801 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt
     1861 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct
     1921 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct
     1981 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga
     2041 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag
     2101 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct
     2161 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg
     2221 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc
     2281 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct
     2341 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga
     2401 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga
     2461 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg
     2521 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt
     2581 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt
     2641 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt
     2701 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc
     2761 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa
     2821 agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt
     2881 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct
     2941 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga
     3001 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac
     3061 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg
     3121 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac
     3181 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca
     3241 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttgtc tggctggcga
     3301 tactctcatt accctggccg atggacgacg agtgcctatt agagaactgg tgtcacagca
     3361 gaatttttcc gtgtgggctc tgaatcctca gacttaccgc ctggagaggg ctagagtgag
     3421 tagagctttc tgtaccggca tcaaacctgt gtaccgcctc accactagac tggggagatc
     3481 cattagggcc actgccaacc accgatttct cacacctcag ggctggaaac gagtcgatga
     3541 actccagcct ggagattacc tggctctgcc taggagaatc cctactgcct cctgatctcg
     3601 agctcgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3661 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3721 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     3781 gcaagtaaaa cctctacaaa tgtggtactt aagaggggga gaccaaaggg cacgcgtcga
     3841 gtccaacacc cgtgggaatc ccatgggcac catggcccct cgctccaaaa atgctttcgc
     3901 gtcgcgcaga cactgctcgg tagtttcggg gatcagcgtt tgagtaagag cccgcgtctg
     3961 aaccctccgc gccgccccgg ccccagtgga aagacgcgca ggcaaaacgc accacgtgac
     4021 ggagcgtgac cgcgcgccga gcgcgcgcca aggtcgggca ggaagagggc ctatttccca
     4081 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     4141 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     4201 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     4261 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgagtctt
     4321 cgaggaagac aggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag
     4381 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctaga gggcggccgc
     4441 aggtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt
     4501 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4561 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc cttaattaaa
     4621 tctggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4681 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4741 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4801 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4861 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4921 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4981 gtgagcaaaa ccgcagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5461 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5641 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttaccatta ctacaggcat
     6121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6481 gcgaaaactc tcaaggatct taccactatt gagatccagt tcgatgtaac ccactcgtgc
     6541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaagat
     6781 gccacctgaa attataaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     6841 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     6901 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     6961 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     7021 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     7081 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     7141 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     7201 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag
     7261 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     7321 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     7381 acgttgtaaa acgacggcca gtgaattagg ttaattaagg ctgcgcgctc gctcgctcac
     7441 tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
     7501 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct tgtagttaat
     7561 gattaacccg ccatgctact tatctacctg c
//
