LOCUS       Exported                7344 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV expressing deactivated S. aureus Cas9 fused to a KRAB repressor.
ACCESSION   .
VERSION     .
KEYWORDS    AAV CMV-dSaCas9-KRAB-bGHpA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7344)
  AUTHORS   Thakore PI, Kwon JB, Nelson CE, Rouse DC, Gemberling MP, Oliver ML, 
            Gersbach CA
  TITLE     RNA-guided transcriptional silencing in vivo with S. aureus 
            CRISPR-Cas9 repressors.
  JOURNAL   Nat Commun. 2018 Apr 26;9(1):1674. doi: 10.1038/s41467-018-04048-4.
  PUBMED    29700298
REFERENCE   2  (bases 1 to 7344)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7344
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     204..223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   365..494
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        518..897
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        898..1101
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1052..1072
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1129..1155
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1159..1179
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4360..4407
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4441..4635
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             4702..4722
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4750..4767)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4756..4963
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4971..5111
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4971..5100
                     /label=AAV2 ITR
     rep_origin      5186..5641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5273..5292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5483..5504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5658..5677)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5777..5799
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5837..5855)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5923..6027
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6028..6888
                     /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     complement(6246..6265)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7059..303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
       61 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      121 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      181 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      241 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      301 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      361 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
      421 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      481 cactaggggt tcctgcggcc tctagactcg aggcgttgac attgattatt gactagttat
      541 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      601 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      661 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      721 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      781 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      841 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      901 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      961 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1021 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1081 gaggtctata taagcagagc tctctggcta actaccggtg ccaccatgta cccatacgat
     1141 gttccagatt acgctgcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     1201 gccaagcgga actacatcct gggcctggcc atcggcatca ccagcgtggg ctacggcatc
     1261 atcgactacg agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac
     1321 gtggaaaaca acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg
     1381 cggcatagaa tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac
     1441 agcgagctga gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg
     1501 agcgaggaag agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac
     1561 gtgaacgagg tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg
     1621 aacagcaagg ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa
     1681 gacggcgaag tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc
     1741 aaacagctgc tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc
     1801 tacatcgacc tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc
     1861 ttcggctgga aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc
     1921 cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac
     1981 gacctgaaca atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag
     2041 ttccagatca tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc
     2101 aaagaaatcc tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag
     2161 cccgagttca ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag
     2221 attattgaga acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc
     2281 agcgaggaca tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc
     2341 gagcagatct ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc
     2401 aacctgatcc tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg
     2461 ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg
     2521 gtggacgact tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg
     2581 atcaacgcca tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc
     2641 gagaagaact ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag
     2701 accaacgagc ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg
     2761 atcgagaaga tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc
     2821 atccctctgg aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc
     2881 agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaagcc
     2941 agcaagaagg gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc
     3001 tacgaaacct tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag
     3061 accaagaaag agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac
     3121 ttcatcaacc ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg
     3181 cggagctact tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc
     3241 accagctttc tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac
     3301 cacgccgagg acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa
     3361 ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc
     3421 atgcccgaga tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc
     3481 aagcacatta aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat
     3541 agagagctga ttaacgacac cctgtactcc acccggaagg acgacaaggg caacaccctg
     3601 atcgtgaaca atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc
     3661 aacaagagcc ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg
     3721 aagctgatta tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa
     3781 accgggaact acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt
     3841 aagtattacg gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc
     3901 agaaacaagg tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat
     3961 ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac
     4021 gaagtgaata gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc
     4081 gagtttatcg cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga
     4141 gtgatcggcg tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacc
     4201 taccgcgagt acctggaaaa catgaacgac aagaggcccc ccaggatcat taagacaatc
     4261 gcctccaaga cccagagcat taagaagtac agcacagaca ttctgggcaa cctgtatgaa
     4321 gtgaaatcta agaagcaccc tcagatcatc aaaaagggca aaaggccggc ggccacgaaa
     4381 aaggccggcc aggcaaaaaa gaaaaaggga tccgatgcta agtcactgac tgcctggtcc
     4441 cggacactgg tgaccttcaa ggatgtgttt gtggacttca ccagggagga gtggaagctg
     4501 ctggacactg ctcagcagat cctgtacaga aatgtgatgc tggagaacta taagaacctg
     4561 gtttccttgg gttatcagct tactaagcca gatgtgatcc tccggttgga gaagggagaa
     4621 gagccctggc tggtggagag agaaattcac caagagaccc atcctgattc agagactgca
     4681 tttgaaatca aatcatcagt tccgaaaaag aaacgcaaag tttaagaatt cctagagctc
     4741 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4801 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4861 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4921 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagggccgc aggaacccct
     4981 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     5041 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     5101 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5161 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5221 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     5281 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5341 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5401 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5461 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5521 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5581 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5641 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5701 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5761 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5821 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5881 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5941 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6001 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6061 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6121 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6181 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6241 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6301 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6361 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6421 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6481 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6541 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6601 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6661 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6721 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     6781 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6841 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6901 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6961 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7021 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7081 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7141 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7201 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7261 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7321 cttaccgggt tggactcaag acga
//
