LOCUS       Exported                7450 bp ds-DNA     circular SYN 17-FEB-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAAV2_SlutCas9_U6_BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7450)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 18, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7450
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    39..130
                     /label=AAV2 ITR
     enhancer        154..533
                     /label=CMV enhancer
     promoter        534..737
                     /label=CMV promoter
     CDS             768..788
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(790..812)
                     /label=SaCas9-R2
     CDS             810..3971
                     /codon_start=1
                     /label=SlutCas9
                     /translation="ARNSYILGLDIGITSVGYGIIDRVTREVIDAGVRLFPEANVENNE
                     GRRSKRGARRLKRRRIHRLNRIKQLLKNAGLLEGDVLPKSTNPYEIRVRGLRSPLTKDE
                     LVIALLHIAKRRGIHNINIVGDDEETDSTLSTTAQLKKNEKALKGQFVCELQLDRLANA
                     HQVRGEKNRFKTEDIVKEVRALLQQQQNFHNIDNSFVEQYIALLESRRTYYEGPGEGSP
                     YGWDGDIKKWYEMLMGYCTYFPEELRSVKYAYTADLFNALNDLNNLVITRDDNSKLTYA
                     EKYHIIENVFKQKKVPTLKQIAKEIGVNESDIKGYRINKSEKPLFTSFKLYHDLKSVFS
                     DPTKLEDIDLLDRIAVVLTMYQDAESMKAALNTFPEVFSEAEKEKLSALTGYAGTHRLS
                     LKCMNLLIPDLWQTSLNQMELFVKLNLKPQKLDLSQCHQIPTQLVDEFILSPVVKRAFT
                     QSIKVINAIIQKYGLPDDIIIELAREKNSADKRKFLNQLQKKNEKARHEINTLVAQYGQ
                     PNAKRLVEKITLHQQQEGKCLYSLKDIPLEQLLKQPYLYEVDHIIPRSVSFDNSMQNKV
                     LVLAEENAKKGNQTPYQYLNSREASMTYPEFKQHILNLSKAKDRISKKKRNYLLEERDI
                     NKFDVQKDFINRNLVDTRYATRELASLLKAYFKTHELPVKVKTINGGFTHYLRKVWKFD
                     KDRNKGYKHHAEDALIIANADFLFKNQTLNKIEAILNEPGREVESDTVKVQSEDNYQDL
                     FENTKKAFAIKNFKDFKFSHRVDQKPNRQLVNDTLYSTREVNEDLYVVQTLKDIYSKDN
                     KDVKRLFDKQPEKFLMFQHDPETFKKFELAMKQYAEEKNPLARYYEEQGYITKYAKKGD
                     GPPVKSLKYIGKKVGKHLDVTGDYEDSNRKLVKLSLKSFRFDIYHTDKGYKMVPITYLD
                     VQKKEKYYYIPTEKYEALKQEKGINQNAQFIGSFYYNDLIEFDGELYRVIGINNGDKNL
                     VELDMVDIRYKEYCELNSITTTPRIVKTISPKTQSIEKYTTDILGNLYKAQPGKKPQFI
                     FNKDED"
     primer_bind     3817..3835
                     /label=qRCR-SlutCas9-F
     primer_bind     complement(3932..3950)
                     /label=qRCR-SlutCas9-R
     CDS             3972..4019
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     3972..3989
                     /label=SaCas9-F
     CDS             4026..4052
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4053..4079
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4080..4106
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4140..4347
                     /label=PolyA
     primer_bind     complement(4158..4177)
                     /label=Primer 6
     promoter        4355..4595
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4624..4704
                     /label=sgRNA
     repeat_region   4713..4853
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4928..5383
                     /direction=RIGHT
                     /label=f1 ori
     promoter        5665..5769
                     /label=AmpR promoter
     CDS             5770..6630
                     /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"
     rep_origin      6801..7389
                     /direction=RIGHT
                     /label=ori
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccagaaacag ctacatcctg ggcctggaca
      841 tcggaatcac cagcgtggga tatggcatca tcgacagagt caccagagag gtgatcgacg
      901 ccggcgtgcg gcttttcccc gaggccaacg tggagaacaa cgagggcaga cggagcaaga
      961 gaggagcccg gcggctgaaa agaaggcgga tccaccggct gaatagaatc aagcagctgc
     1021 tgaaaaacgc cggcctgctg gagggagatg tgctgcctaa gtctaccaac ccctatgaga
     1081 tcagagtgcg gggcctccga agccctctga ccaaagatga actggtgatc gccctgctcc
     1141 acatcgccaa aagaagaggc atccacaaca tcaacatcgt gggagatgac gaagaaacgg
     1201 acagcacact gagtaccaca gcccagctga agaagaacga gaaggctctc aagggacagt
     1261 ttgtttgtga actgcaactg gacagactgg ctaatgccca ccaagtgcgg ggcgagaaaa
     1321 atcgatttaa gacagaggac attgtgaagg aagtcagagc cctgcttcag caacagcaga
     1381 acttccacaa catcgataat tctttcgtgg aacagtacat tgccctgctg gagagccgga
     1441 ggacctacta cgagggccct ggcgaaggct ctccttacgg ctgggacggc gacattaaga
     1501 agtggtacga gatgctgatg ggctattgca cctacttccc tgaagagctg agaagcgtga
     1561 agtacgccta caccgccgat ctgttcaacg ccctgaatga cctgaacaac ctggtgatca
     1621 cccgggacga caacagcaaa ttgacatacg ccgagaagta ccatatcatc gagaacgtgt
     1681 tcaaacagaa gaaagtacct acactgaagc agatcgccaa ggaaatcgga gtgaacgaga
     1741 gcgatattaa gggctacaga atcaacaaat ctgagaaacc tctgttcacc agcttcaaac
     1801 tgtaccacga tctgaagagc gtgttcagcg accctacaaa actggaagat atcgacttgc
     1861 tggaccgaat cgccgtggtg ctgaccatgt accaagatgc cgaatccatg aaagccgccc
     1921 tgaacacctt ccctgaagtg ttcagcgaag cagagaaaga gaagctgagc gccctcacag
     1981 gctacgctgg cacccataga ctgtctctga agtgcatgaa cctgctgatc cctgatctgt
     2041 ggcagacaag cctgaaccag atggaactgt tcgtgaagct gaatctgaaa ccacagaagc
     2101 tggacctgag ccagtgccac cagattccta cccagctggt ggacgagttc atcctgtctc
     2161 ctgtggtgaa aagagccttc acccaaagca tcaaggtgat caacgccatc atccagaaat
     2221 acggcctgcc cgacgacatc ataatcgagc tggccaggga aaaaaacagc gccgataagc
     2281 ggaagttcct gaatcagctg cagaagaaga acgagaaggc ccggcacgag atcaataccc
     2341 tggtggccca gtacggccag ccaaatgcta agcggctggt ggaaaagatc acactgcacc
     2401 agcagcagga gggcaagtgt ctgtactccc tgaaggatat ccccctggag cagctgctga
     2461 agcagcccta cctgtacgag gtggaccaca tcatccccag aagcgtttct ttcgacaaca
     2521 gcatgcagaa caaggtgctg gtcctggccg aagaaaacgc caaaaagggc aaccagaccc
     2581 cttaccagta cctgaatagc agagaggcca gcatgaccta ccccgaattc aaacagcaca
     2641 tcctgaatct gagcaaggcc aaggaccgga tcagcaagaa gaagcggaac tacctgctcg
     2701 aggaaagaga tatcaacaag ttcgacgtgc aaaaggactt catcaacaga aacctggttg
     2761 acaccagata cgccaccaga gagctcgcct ctctgctgaa ggcttatttc aagacacatg
     2821 agctccctgt gaaagtgaag accatcaacg gcggatttac ccactacctg agaaaggtgt
     2881 ggaagtttga caaagataga aacaagggct acaagcacca cgccgaggat gcactgatca
     2941 tcgccaacgc cgactttctg tttaagaacc agactctgaa caaaatcgag gctatcctga
     3001 acgagcccgg cagagaggtg gaatctgaca cagtgaaggt gcagagcgag gacaattacc
     3061 aggacctgtt tgagaacacc aagaaggctt tcgccatcaa gaatttcaag gatttcaagt
     3121 ttagccacag agtggaccag aagcctaacc ggcagctcgt gaacgacacc ctgtacagca
     3181 ccagagaggt gaacgaagat ctgtacgtgg tgcagaccct gaaggacatc tacagcaagg
     3241 acaacaaaga cgtgaagcgg ctcttcgaca agcaacccga gaagttcctg atgttccagc
     3301 acgaccccga gacattcaag aaattcgagc tggctatgaa gcaatatgcc gaagagaaga
     3361 accctctggc tagatactac gaggaacagg gctacatcac caagtacgcc aagaaaggag
     3421 acggcccacc tgtgaaaagc ctcaaataca tcggcaagaa ggtgggaaaa cacctggatg
     3481 tgaccggcga ctacgaggat agcaaccgga agctggtgaa gctgagcctg aagtccttta
     3541 gattcgatat ctaccacaca gacaagggct acaagatggt tccaatcact tacctggatg
     3601 tgcagaaaaa agagaagtac tactacatcc ccaccgagaa atacgaagcc ctgaagcagg
     3661 agaagggcat caaccagaat gctcagttca ttggcagctt ctactacaac gacctgatcg
     3721 agttcgatgg cgagctgtat agagtgatcg gcatcaacaa cggcgacaag aatctcgttg
     3781 aactcgacat ggtggacatt agatataagg aatactgcga gctgaactcc atcaccacca
     3841 cacctagaat cgttaagacc atcagcccca agacccagag catcgagaag tacacaacag
     3901 atatcctggg caacctgtac aaagcccagc ctggcaagaa gcctcagttc atcttcaaca
     3961 aagacgagga taaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     4021 gatcctaccc atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt
     4081 acccatacga tgttccagat tacgcttaag aattcctaga gctcgctgat cagcctcgac
     4141 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4201 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4261 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4321 ggaagagaat agcaggcatg ctggggaggt acctgagggc ctatttccca tgattccttc
     4381 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     4441 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     4501 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     4561 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacc acggcaggtc
     4621 tcagttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct
     4681 cgtcaacttg ttggcgagat ttttgcggcc gcaggaaccc ctagtgatgg agttggccac
     4741 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4801 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4861 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4921 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     5161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     5281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5341 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5401 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5461 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5521 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5581 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5641 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5701 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5761 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5821 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5881 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5941 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6001 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6061 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6121 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6181 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6241 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6301 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6361 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6421 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6481 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6541 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6601 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6661 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6721 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6781 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6841 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6901 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6961 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7021 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7081 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7141 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7201 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7261 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7321 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7381 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7441 gctcacatgt
//
