LOCUS       sequence-228612-        7930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV Vector for expression of C-terminal SpyCas9 fragement with 
            split-intein and a CMV-driven AcrIIA4 with two miR-122 binding 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7930)
  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 7930)
  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-AcrIIA4-2xmiR-122 ta...
FEATURES             Location/Qualifiers
     source          1..7930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        263..642
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        643..846
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          938..1070
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1100..1109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1104..1113
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1710..1721
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3239..3259
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3270..3289)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3299..3380
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3324..3343
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3478..3857
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3858..4061
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4106..4125
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4106..4124
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4524..4541)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4530..4754
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4808..4937
                     /label=AAV2 ITR
     primer_bind     complement(4975..4991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4975..4991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4988..5010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4999..5015
                     /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(5023..5029,5030..5047,5048..5053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5068..5089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5377..5965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5457..5476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6136..6927,6928..6996))
                     /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     6759..6778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6997..7101)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7128..7583)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7265..7286)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7477..7496
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7710..7732
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7724..7741
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7725..7741
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7760..7889
                     /label=AAV2 ITR
ORIGIN
        1 ggccgccatt atggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac cagtcccggt ctcctcccat
      121 gcatgtcaat attggccatt agccatatta ttcattggtt atatagcata aatcaatatt
      181 ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta tattggctca
      241 tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt
      301 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      361 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      421 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      481 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc
      541 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct
      601 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      661 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      721 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat
      781 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      841 agaggtcgtt tagtgaaccg tcagatcact agtagcttta ttgcggtagt ttatcacagt
      901 taaattgcta acgcagtcag tgctcgactg atcacaggta agtatcaagg ttacaagaca
      961 ggtttaagga ggccaataga aactgggctt gtcgagacag agaagattct tgcgtttctg
     1021 ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag gggtaccgaa
     1081 gccgctagcg ctaccggtcg ccaccatggc ggcggcgtgc ccggaactgc gtcagctggc
     1141 gcagagcgat gtgtattggg atccgattgt gagcattgaa ccggatggcg tggaagaagt
     1201 gtttgatctg accgtgccgg gcccgcataa ctttgtggcg aacgatatta ttgcgcataa
     1261 ctctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat
     1321 caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag
     1381 gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg
     1441 acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc
     1501 ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct
     1561 gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct
     1621 ctccgactac gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga
     1681 taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga
     1741 agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac
     1801 acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa
     1861 agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca
     1921 aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt
     1981 gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta
     2041 taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt
     2101 aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta
     2161 taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac
     2221 cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc
     2281 caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt
     2341 gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa
     2401 catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa
     2461 aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg
     2521 attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa
     2581 gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag
     2641 cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga
     2701 cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat
     2761 gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt
     2821 taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga
     2881 gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat
     2941 aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc
     3001 ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt
     3061 tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag
     3121 aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac
     3181 ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc
     3241 caagaagaag aggaaggtgt ctcgagctcg atgagtttgg acaaaccaca actagaatgc
     3301 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3361 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3421 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atctagagac
     3481 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     3541 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     3601 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     3661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3781 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3841 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     3901 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     3961 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     4021 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     4081 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     4141 agcatgaaca ttaacgacct gattcgggaa ataaagaaca aggactacac ggtcaagctg
     4201 tcaggcaccg actccaactc tataacacag ctcattataa gggttaataa tgatggcaac
     4261 gaatatgtca tctctgaatc cgagaatgaa tcaatcgtgg agaaattcat cagtgcattt
     4321 aaaaacggct ggaaccagga gtacgaagat gaggaagagt tctacaatga tatgcaaact
     4381 atcacactga aatctgagct gaactagctc gagcggccgc cactgtgctg gatatctgca
     4441 gaattacaaa caccattgtc acactccatt atatacaaac accattgtca cactccatag
     4501 cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4561 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4621 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4681 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4741 ggtgggctct atggacgcgt gcggccgcag gtagataagt agcatggcgg gttaatcatt
     4801 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4861 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4921 agcgagcgag cgcgcagcct aattaaggcc ttaattaaat ctggcgtaat catggtcata
     4981 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5041 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5101 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5161 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaac cgcagcaaaa
     5341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6241 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6421 taatagtttg cgcaacgttg ttaccattac tacaggcatc gtggtgtcac gctcgtcgtt
     6481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6841 accactattg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7081 taaacaaata ggggttccgc gcacatttcc ccgaaagatg ccacctgaaa ttataaacgt
     7141 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     7201 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     7261 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     7321 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     7381 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     7441 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     7501 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     7561 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     7621 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     7681 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     7741 tgaattaggt taattaaggc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
     7801 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     7861 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
     7921 atctacctgc
//
