LOCUS       Exported                8140 bp ds-DNA     circular SYN 14-OCT-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAAV2_SauriCas9_P2A_Puro_U6_BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8140)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 6, 2019 from SnapGene Viewer 5.0.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8140
                     /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..3992
                     /codon_start=1
                     /label=SauriCas9-KKH
                     /translation="AQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRLFPEAD
                     SENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQMIDLNNVPKSTDPYTIRVKGLREP
                     LTKEEFAIALLHIAKRRGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCELQLE
                     RLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNIDDQFIQQYIDLVSTRREYFEGP
                     GNGSPYGWDGDLLKWYEKLMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDNP
                     KLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRGYRITKSGKPQFTSFKLYHDL
                     KNIFEQAKYLEDVEMLDEIAKILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTG
                     THRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSEIDSIPTTLVDEFILSPVV
                     KRAFIQSIKVINAVINRFGLPEDIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLL
                     AKYGNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTHYEVDHIIPRSVSFDNS
                     LNNKVLVKQSENSKKGNRTPYQYLSSNESKISYNQFKQHILNLSKAKDRISKKKRDMLL
                     EERDINKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVKVKTINGGFTNHLRK
                     VWDFKKHRNHGYKHHAEDALVIANADFLFKTHKALRRTDKILEQPGLEVNDTTVKVDTE
                     EKYQELFETPKQVKNIKQFRDFKYSHRVDKKPNRKLINDTLYSTREIDGETYVVQTLKD
                     LYAKDNEKVKKLFTERPQKILMYQHDPKTFEKLMTILNQYAEAKNPLAAYYEDKGEYVT
                     KYAKKGNGPAIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFDIYKCEQGYKM
                     VSIGYLDVLKKDNYYYIPKDKYEAEKQKKKIKESDLFVGSFYKNDLIMYEDELFRVIGV
                     NSDINNLVELNMVDITYKDFCEVNNVTGEKHIKKTIGKRVVLIEKYTTDILGNLYKTPL
                     PKKPQLIFKRGEL"
     primer_bind     2496..2515
                     /label=R5-BglII-F
     primer_bind     complement(3152..3189)
                     /label=R5-788-R
     primer_bind     3171..3189
                     /label=R5-788-F
                     /note="Gln788Lys"
     unsure          3171..3173
                     /label=Gln788Lys
     primer_bind     complement(3705..3744)
                     /label=R5-973-R
     primer_bind     3726..3747
                     /label=R5-973-F
                     /note="Tyr973Lys"
     unsure          3726..3728
                     /label=Tyr973Lys
     primer_bind     complement(3848..3869)
                     /label=R5-1020-R
                     /note="Arg1020His"
     primer_bind     3852..3889
                     /label=R5-1020-F
     unsure          3867..3869
                     /label=Arg1020His
     CDS             3993..4040
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     3993..4010
                     /label=SaCas9-F
     primer_bind     4026..4064
                     /label=Gibsin-P2A-F
     misc_feature    4053..4118
                     /label=P2A
     misc_feature    4119..4715
                     /label=Puro
                     /label=Puro(-tga)
     primer_bind     complement(4583..4615)
                     /label=Puro-R
     primer_bind     4598..4619
                     /label=Puro-F
     primer_bind     complement(4698..4734)
                     /label=Gibson-puro-R
     CDS             4716..4742
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4743..4769
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4770..4796
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4830..5037
                     /label=PolyA
     primer_bind     complement(4848..4867)
                     /label=polyA-R
     promoter        5045..5285
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    5314..5394
                     /label=sgRNA
     repeat_region   5403..5543
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5618..6073
                     /direction=RIGHT
                     /label=f1 ori
     promoter        6355..6459
                     /label=AmpR promoter
     CDS             6460..7320
                     /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      7491..8079
                     /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 cccaggagaa ccagcagaag cagaactaca
      841 tcctgggcct ggacatcgga atcaccagcg tcggctacgg actgatcgat agcaagacaa
      901 gagaagtgat cgacgccggc gttagactct ttccagaagc tgatagcgag aacaactcca
      961 accgcagaag caagcggggc gccagacggt taaaacggag aagaatccac cggctgaacc
     1021 gggtcaaaga cctgctcgct gattaccaga tgatcgatct taacaatgtt cctaagagca
     1081 ccgaccccta caccatcaga gtgaagggcc tccgggagcc tctgacaaaa gaagaattcg
     1141 ccatcgccct cctgcatatc gctaagagaa gaggcctgca caacatcagt gtgtccatgg
     1201 gcgacgaaga gcaggacaat gaactgagca ccaagcagca gctgcaaaag aatgcccagc
     1261 aactgcagga caagtatgtg tgcgaactgc agttagaacg gctgaccaac atcaacaagg
     1321 tcagaggcga gaagaacaga tttaagacag aggactttgt gaaagaagtg aaacagctgt
     1381 gcgaaaccca gagacagtac cacaacatcg acgaccaatt catccagcag tacatcgacc
     1441 tggtgtctac aagacgggag tacttcgagg gccccggcaa cggctctcca tacggctggg
     1501 acggcgacct gctgaagtgg tacgagaagc tgatgggcag atgcacctat ttccccgaag
     1561 aactgaggtc cgtgaagtac gcctacagcg ccgacctctt caacgccctg aacgacctga
     1621 acaacctcgt tgtgaccagg gatgacaatc caaagcttga gtactacgag aagtaccaca
     1681 ttattgagaa cgtgttcaag caaaagaaga atcccacact caaacaaatc gccaaagaga
     1741 tcggcgtgca agattacgac atccggggct atagaatcac aaagagcggc aaacctcagt
     1801 tcacctcttt taagctgtat cacgacctga agaacatctt cgagcaggcc aaatacctgg
     1861 aagatgtgga aatgctggac gagatcgcca agatcctgac catctaccag gatgagatta
     1921 gcatcaagaa agccctggac cagctgcccg aactgctgac agagagcgag aaatctcaga
     1981 tcgcacagct caccggctat acaggcaccc acagactgag cctgaagtgc atccacattg
     2041 tgatcgacga gctgtgggag agccccgaga accagatgga aatctttacc agactgaatc
     2101 tgaaacctaa gaaggtggaa atgagcgaga tcgacagcat acccaccacc ctggtcgacg
     2161 agttcatcct ctcacctgtg gtgaagcggg ccttcatcca gagcatcaag gtaatcaacg
     2221 cagtgatcaa tcggttcggc ctgccagagg acatcatcat cgagctggcc agagaaaaga
     2281 atagcaagga tcggagaaag ttcattaaca agctgcagaa acaaaatgag gccacaagaa
     2341 agaaaatcga acagctgctg gccaagtacg gcaacaccaa tgccaagtac atgatcgaga
     2401 agatcaagct gcacgacatg caggagggca agtgcctgta cagcctggag gctattcctc
     2461 tggaagacct gctgagcaac ccgacacact acgaagttga ccacattatc cccagatctg
     2521 tgagctttga caacagcctg aacaacaaag tgctggtgaa acaaagcgaa aacagcaaga
     2581 agggcaatcg caccccttac cagtacctga gcagcaacga gtctaagatt agctacaacc
     2641 agtttaagca gcacatcctg aacctgagca aggccaagga cagaatcagc aagaaaaaaa
     2701 gagatatgct gctggaagag agagatatca acaagttcga agtgcagaag gaattcatta
     2761 accggaacct ggtggataca cggtacgcca ccagagaact gtctaacctg ctgaagacct
     2821 acttcagcac ccatgactac gccgtgaagg tgaagaccat caacggcggc ttcactaacc
     2881 acctgaggaa ggtgtgggat ttcaagaagc acagaaacca cggctacaag caccacgccg
     2941 aagatgccct ggtgatcgcc aacgccgact tcctgtttaa gacacataag gccctgcgga
     3001 gaaccgataa gatcctggaa caacctggcc tggaagtgaa tgatacaacc gtgaaagtgg
     3061 acaccgagga aaaataccag gagctgttcg agacacctaa gcaagtgaag aacatcaagc
     3121 agttccggga cttcaagtac agccaccgag tggacaagaa gcctaaccgg aagcttatca
     3181 acgacacact gtactccacc agagagattg atggcgaaac ctacgtggtg cagaccctta
     3241 aggatctgta cgccaaggac aacgagaaag tgaagaagct gttcaccgaa agacctcaga
     3301 agatcctgat gtaccagcac gaccctaaga ccttcgagaa actgatgaca atcctgaacc
     3361 agtacgctga ggccaagaac cctctggctg cttattacga ggacaaaggc gagtacgtga
     3421 ccaagtacgc caagaaaggc aatggacctg ccatccacaa gatcaagtat atcgataaga
     3481 agcttggatc ttacctggat gttagcaaca agtatcctga gacacagaac aagcttgtga
     3541 agctgtccct gaagagcttt agattcgaca tctacaagtg tgaacagggc tacaagatgg
     3601 tgtccatcgg atacctggac gtgctgaaga aagataacta ctactacatc cctaaggaca
     3661 agtacgaggc cgagaagcag aaaaagaaga tcaaggaatc tgatcttttt gtgggcagct
     3721 tctacaagaa cgacctcatc atgtacgagg atgaactgtt cagagtgata ggagtgaaca
     3781 gcgacatcaa caatctggtt gagctaaaca tggtcgacat tacctacaag gacttctgcg
     3841 aggtgaacaa cgtgacaggc gagaaacaca tcaaaaagac tatcggcaag cgcgtggtcc
     3901 tgatcgagaa gtacaccaca gatattctag gcaacctgta caagactccc ctgcctaaga
     3961 agccccagct tatcttcaag cggggagaac tgaaaaggcc ggcggccacg aaaaaggccg
     4021 gccaggcaaa aaagaaaaag ggatccgcta gcggcagcgg cgccaccaac ttcagcctgc
     4081 tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gaccgagtac aagcccacgg
     4141 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     4201 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     4261 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     4321 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     4381 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     4441 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     4501 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     4561 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc
     4621 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     4681 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaccc atacgatgtt ccagattacg
     4741 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttaag
     4801 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4861 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4921 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4981 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
     5041 acctgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     5101 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     5161 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     5221 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     5281 aggacgaaac accggagacc acggcaggtc tcagttttag tactctggaa acagaatcta
     5341 ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat ttttgcggcc
     5401 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5461 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5521 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5581 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5641 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     5701 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5761 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5821 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5881 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5941 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     6001 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6061 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6121 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6181 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6241 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6301 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6421 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6481 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6541 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6601 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6661 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6721 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6781 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6841 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6901 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6961 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7021 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7081 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7141 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     7201 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7261 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7321 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7381 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     7681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8101 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
