LOCUS       Exported                7929 bp ds-DNA     circular SYN 13-MAY-2021
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    AAV Intein-C-SpyCas9, CMV-AcrIIA4-2xmiR-122 target sites
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7929
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          296..428
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      458..467
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      462..471
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1068..1079
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2597..2617
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2628..2647)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    2657..2738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2682..2701
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        2836..3215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3216..3419
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3464..3483
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3464..3482
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3882..3899)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3888..4112
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4166..4295
                     /label=AAV2 ITR
     primer_bind     complement(4333..4349)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4333..4349)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4346..4368)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4357..4373
                     /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(4381..4411)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4426..4447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4564..4581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4735..5323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4815..4834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5494..6354)
                     /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     6117..6136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6355..6459)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6485..6940)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6622..6643)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6834..6853
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7067..7089
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7081..7098
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7082..7098
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7117..7246
                     /label=AAV2 ITR
     primer_bind     complement(7300..7320)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        7550..7929
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag aggtcgttta gtgaaccgtc agatcactag tagctttatt
      241 gcggtagttt atcacagtta aattgctaac gcagtcagtg ctcgactgat cacaggtaag
      301 tatcaaggtt acaagacagg tttaaggagg ccaatagaaa ctgggcttgt cgagacagag
      361 aagattcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct
      421 ctccacaggg gtaccgaagc cgctagcgct accggtcgcc accatggcgg cggcgtgccc
      481 ggaactgcgt cagctggcgc agagcgatgt gtattgggat ccgattgtga gcattgaacc
      541 ggatggcgtg gaagaagtgt ttgatctgac cgtgccgggc ccgcataact ttgtggcgaa
      601 cgatattatt gcgcataact ctggccaggg ggacagtctt cacgagcaca tcgctaatct
      661 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
      721 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
      781 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
      841 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
      901 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
      961 actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct
     1021 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag
     1081 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     1141 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     1201 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     1261 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     1321 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     1381 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     1441 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga
     1501 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     1561 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     1621 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     1681 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct
     1741 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     1801 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     1861 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     1921 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     1981 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata
     2041 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga
     2101 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     2161 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     2221 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     2281 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct
     2341 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     2401 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     2461 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact
     2521 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     2581 agacagcagg gctgacccca agaagaagag gaaggtgtct cgagctcgat gagtttggac
     2641 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     2701 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     2761 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     2821 aatgtggtat ctagagacat tgattattga ctagttatta atagtaatca attacggggt
     2881 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2941 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     3001 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     3061 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     3121 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     3181 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     3241 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     3301 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     3361 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     3421 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     3481 gggagaccca agctggctag catgaacatt aacgacctga ttcgggaaat aaagaacaag
     3541 gactacacgg tcaagctgtc aggcaccgac tccaactcta taacacagct cattataagg
     3601 gttaataatg atggcaacga atatgtcatc tctgaatccg agaatgaatc aatcgtggag
     3661 aaattcatca gtgcatttaa aaacggctgg aaccaggagt acgaagatga ggaagagttc
     3721 tacaatgata tgcaaactat cacactgaaa tctgagctga actagctcga gcggccgcca
     3781 ctgtgctgga tatctgcaga attacaaaca ccattgtcac actccattat atacaaacac
     3841 cattgtcaca ctccatagct taagtttaaa ccgctgatca gcctcgactg tgccttctag
     3901 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3961 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4021 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4081 caggcatgct ggggatgcgg tgggctctat ggacgcgtgc ggccgcaggt agataagtag
     4141 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     4201 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4261 gcccgggcgg cctcagtgag cgagcgagcg cgcagcctaa ttaaggcctt aattaaatct
     4321 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4381 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4441 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4501 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4561 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4621 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4681 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4741 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4801 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4861 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4921 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4981 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5041 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5101 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5161 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5221 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5281 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5341 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5401 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5461 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5521 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5581 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5641 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5701 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5761 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5821 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5881 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5941 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6001 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6061 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6121 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6181 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6241 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6301 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6361 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6421 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6481 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     6541 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     6601 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     6661 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     6721 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     6781 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     6841 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     6901 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     6961 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     7021 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     7081 tgtaaaacga cggccagtga attaggttaa ttaaggctgc gcgctcgctc gctcactgag
     7141 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     7201 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttccttgta gttaatgatt
     7261 aacccgccat gctacttatc tacctgcggc cgccattatg gaatgtgtgt cagttagggt
     7321 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     7381 cagcaaccag tcccggtctc ctcccatgca tgtcaatatt ggccattagc catattattc
     7441 attggttata tagcataaat caatattggc tattggccat tgcatacgtt gtatctatat
     7501 cataatatgt acatttatat tggctcatgt ccaatatgac cgccatgttg gcattgatta
     7561 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     7621 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     7681 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     7741 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     7801 atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     7861 cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     7921 attaccatg
//
