LOCUS       sequence-232735-        7929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV Vector for expression of C-terminal SpyCas9 fragemnt with 
            split-intein and a CMV-driven AcrIIA4 (no miR binding sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7929)
  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 7929)
  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 Intein-C-SpyCas9, CMV-AcrIIA4-scaffold (2x...
FEATURES             Location/Qualifiers
     source          1..7929
                     /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"
     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"
     regulatory      842..851
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1448..1459
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2977..2997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3008..3027)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3037..3118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3062..3081
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3216..3595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3596..3799
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3844..3863
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3844..3862
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4262..4279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4268..4492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4546..4675
                     /label=AAV2 ITR
     primer_bind     complement(4713..4729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4713..4729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4726..4748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4737..4753
                     /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(4761..4767,4768..4785,4786..4791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4806..4827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5115..5703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5195..5214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5874..6665,6666..6734))
                     /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     6497..6516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6735..6839)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6865..7320)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7002..7023)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7214..7233
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7447..7469
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7461..7478
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7462..7478
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7497..7626
                     /label=AAV2 ITR
     primer_bind     complement(7680..7700)
                     /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 accatggcgg cggcgtgccc ggaactgcgt cagctggcgc agagcgatgt gtattgggat
      901 ccgattgtga gcattgaacc ggatggcgtg gaagaagtgt ttgatctgac cgtgccgggc
      961 ccgcataact ttgtggcgaa cgatattatt gcgcataact ctggccaggg ggacagtctt
     1021 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     1081 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     1141 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     1201 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     1261 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     1321 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat
     1381 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     1441 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     1501 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     1561 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     1621 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     1681 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     1741 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     1801 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     1861 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     1921 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     1981 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     2041 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     2101 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     2161 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     2221 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
     2281 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     2341 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     2401 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     2461 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
     2521 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     2581 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
     2641 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
     2701 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
     2761 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
     2821 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
     2881 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
     2941 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtct
     3001 cgagctcgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3061 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3121 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     3181 aagcaagtaa aacctctaca aatgtggtat ctagagacat tgattattga ctagttatta
     3241 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     3301 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     3361 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     3421 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     3481 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     3541 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     3601 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     3661 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     3721 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     3781 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     3841 aattaatacg actcactata gggagaccca agctggctag catgaacatt aacgacctga
     3901 ttcgggaaat aaagaacaag gactacacgg tcaagctgtc aggcaccgac tccaactcta
     3961 taacacagct cattataagg gttaataatg atggcaacga atatgtcatc tctgaatccg
     4021 agaatgaatc aatcgtggag aaattcatca gtgcatttaa aaacggctgg aaccaggagt
     4081 acgaagatga ggaagagttc tacaatgata tgcaaactat cacactgaaa tctgagctga
     4141 actagctcga gcggccgcca ctgtgctgga tatctgcaga attagagacg ccgttgatgt
     4201 tgaagtttat ataccgttga tgttgaagtc gtctctagct taagtttaaa ccgctgatca
     4261 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4321 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4381 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4441 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggacgcgtgc
     4501 ggccgcaggt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     4561 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4621 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagcctaa
     4681 ttaaggcctt aattaaatct ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4741 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4801 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4861 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4921 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4981 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5041 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5101 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5161 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5221 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5281 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5341 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5401 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5461 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5521 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5581 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5641 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5701 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5761 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5821 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5881 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5941 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6001 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6061 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6121 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6181 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6241 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6301 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6361 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6421 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6481 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6541 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6601 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6661 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6721 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6781 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6841 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6901 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6961 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     7021 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     7081 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     7141 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     7201 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     7261 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     7321 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7381 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     7441 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attaggttaa ttaaggctgc
     7501 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc
     7561 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     7621 gttccttgta gttaatgatt aacccgccat gctacttatc tacctgcggc cgccattatg
     7681 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     7741 aagcatgcat ctcaattagt cagcaaccag tcccggtctc ctcccatgca tgtcaatatt
     7801 ggccattagc catattattc attggttata tagcataaat caatattggc tattggccat
     7861 tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac
     7921 cgccatgtt
//
