LOCUS       Exported                7216 bp ds-DNA     circular SYN 04-JAN-2021
DEFINITION  All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    15-Nme2Cas9_AAV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7216)
  AUTHORS   Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, 
            Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
  TITLE     A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo 
            Genome Editing.
  JOURNAL   Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003.
  PUBMED    30581144
REFERENCE   2  (bases 1 to 7216)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Sep 16, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7216
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..21
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             28..75
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(51..75)
                     /label=nme2Cas9-R
     CDS             76..3273
                     /codon_start=1
                     /label=32-nme2
                     /translation="KTPAHRYILGLDLGIASVGWALVLADENEYPIGLLDCGVRVFERA
                     EMPKDGKSLAATRRQARSIRRLIRRRAHRLLRLRRLLKREGLLSAADFAADGLPAAMPV
                     NTWQLRVEGLDRKLAEKEWAAVLLHLVKHRGYLSQRKNEMQTEDKELGKLLAGVAENRQ
                     LLADNPEQYRTPAELAVKRFAVQSGHLRNKGGDYAHTFNRLDLQKELHLLFAKQRGLGN
                     PFASAELENKVDSLLMTQRAALQDGAVLKMLGHCTFEPQEYKAAKHTYSAERFIWLTRL
                     NNLRILHNGEERALNEHERKLLLDEPYKKAKLTYKQVRKILNLPDSAVFKGLRYGKSED
                     GLKAESGALMEMKAYHKIRTVLEKAGLKGEWENRIRNPEVLDAIGTAFSLYKTDNDIQT
                     ALADLALPEKVLEALLKGIGFDKFIQLSLSALRKILPLMEQGLRYDEACTKVYGNHYGA
                     KIQQNSLVLPVIDGNDIRNPVVLRTLTQARKVVNAVIRRYGSPLRVHIETARELGKSWD
                     DRKEIEKRQEENRKERERAADEFKEYFPHFAGEPKGRDILKMRLYNQQQGKCLYSGLPL
                     DLSRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQKKRSQTPYEYLDGASNSERW
                     REFQARVAGCHFPYAKKQRIQTAKLNEQDFIDRNLNDTRYIARFLCGFIENNLHLEGKD
                     KRRVFASNGQITALLRGLWGLRKVREENDRHHALDAVVVACSTVSMQQKITEAMKRRES
                     LETVNTETGEVKQRIPQPWDFFRQEVMIRVFSDHPREELAEKLNSRPQALHEHVAPLFV
                     SRAPNRKTTGQGHLETIKSAKRLSENISVVKKPLASLKTKDIENIAGYTANREPEFYQA
                     LKKRLAEFNDDPKKAFAEPFYKPTKNGEQGPLVKSVRVEDDRVKVGLAVRHGITDTNGD
                     MVRADVYQKANKSGRIQYFVVPVYAWQIAKGVLPLLDCRGYKIDETYTFCFSLHKYDLI
                     SFPKDGNPEEEQLVYYITCDSSNGRFWIASHDKGAKEQQFRISTQNQLFIKKYQTDPLG
                     KEIRLCKPEQRPALKIKR"
     CDS             3274..3321
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     3274..3291
                     /label=nme2Cas9-F
     primer_bind     3274..3285
                     /label=Nme2PI-F
     CDS             3322..3411
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3418..3444
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(3450..3469)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      3515..3570
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     repeat_region   3583..3723
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3583..3712
                     /label=AAV2 ITR
     primer_bind     3727..3745
                     /label=ORI-F
     rep_origin      3798..4253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4270..4289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4389..4411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4449..4467)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4535..4639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4640..5500
                     /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(4858..4877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5671..6259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6160..6179
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6295..6312)
                     /label=ORI-R
     repeat_region   6321..6450
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(6478..6570)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(6629..6869)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(6679..6698)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6849..6869)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      7205..7214
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
ORIGIN
        1 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
       61 gccaagaaaa agaaaaaaac ccctgcacac cggtacatcc tgggcctgga tctgggaatc
      121 gccagcgtgg gctgggccct ggtactggcc gacgagaatg aatatcctat cggcctgctg
      181 gattgcggcg tcagggtgtt cgagcgcgct gagatgccca aggacggaaa aagccttgcc
      241 gccacacgta gacaggcccg ctcgatcagg cggctgatcc gtagacgggc ccatagactg
      301 cttagactga gacggctgct gaagcgagaa ggcctcctgt ctgctgctga tttcgccgct
      361 gatggcctgc cggctgccat gcctgttaat acctggcagc tgagagtgga aggcctggac
      421 cgtaagctgg ccgaaaaaga gtgggccgcg gtgctgctgc atctggtcaa gcacagagga
      481 tacctgagcc agagaaagaa cgagatgcag accgaagata aggaactggg aaagctgctg
      541 gctggcgtgg ccgaaaatcg gcagctgctg gccgataacc ccgagcagta cagaacccct
      601 gccgagctgg ctgtgaagag gttcgccgtg cagagcggac acctgagaaa caagggtgga
      661 gattacgccc atacattcaa cagactagac ctgcagaagg aactgcacct cctgttcgcc
      721 aaacagcggg gactgggaaa tcctttcgcc tctgctgagc ttgagaacaa ggtcgactct
      781 ctgctgatga cccagagagc tgccctgcag gacggcgccg tgctgaaaat gctgggtcac
      841 tgcacctttg agccccaaga gtacaaggcc gccaaacaca cctacagcgc tgaaagattt
      901 atctggctga cccggctgaa caacctgcgg atcctgcaca acggagagga gcgggcgctg
      961 aacgagcacg agagaaagct gctgctggat gagccctaca agaaggccaa gctgacatac
     1021 aagcaggtgc ggaagatcct gaatctgccc gattctgccg tgttcaaggg cctgagatac
     1081 ggcaagagcg aggacggtct gaaggccgaa tctggcgccc tcatggaaat gaaagcctac
     1141 cacaagatcc ggaccgtgtt ggagaaggcc ggactgaagg gcgagtggga gaaccggatc
     1201 agaaatcccg aagttctgga cgccatcggc accgccttca gcctgtacaa aaccgacaac
     1261 gacatccaga ccgccctggc tgacctggca cttccagaaa aggtgctgga agccctgctg
     1321 aagggcatcg gcttcgacaa gttcatccag ctgagcctga gcgccctgcg aaagattctg
     1381 cctctgatgg aacaaggcct gcggtatgac gaagcctgta caaaggtgta cggcaaccac
     1441 tacggcgcta agattcagca gaatagcctg gtgctgcctg tgatcgatgg aaacgatatc
     1501 cggaatccag tggtgctgag aacactgacc caggccagaa aggtggtcaa cgccgtgatc
     1561 agaagatacg gcagccctct gagagtgcac atcgagacag ccagagaact gggaaaaagc
     1621 tgggacgata gaaaagagat tgagaagcgg caagaggaga accggaaaga gagagaaaga
     1681 gccgcagatg aattcaaaga atacttccct cactttgccg gcgaacctaa gggaagagat
     1741 atcctgaaaa tgagactgta caaccagcag cagggcaagt gcctgtacag cggcctgcct
     1801 ctggatctgt cccggctcaa cgagaagggc tacgtggaaa tcgaccacgc tctgcctttt
     1861 tctagaacct gggatgacag cttcaataac aaggtgctgg tgctgggcag cgaaaatcag
     1921 aaaaagagat cccagacacc ctacgagtac ctggacggcg cctccaacag cgagcggtgg
     1981 cgggaatttc aggccagagt ggccggctgc cactttcctt atgccaagaa gcaaagaatc
     2041 cagaccgcca aactgaacga gcaggacttc atcgaccgga acctgaacga caccaggtac
     2101 atcgcgcggt tcctgtgcgg cttcatcgag aacaacctgc atctggaagg caaggacaag
     2161 agaagagtgt tcgccagcaa cggccagatc accgcactgc tgcgcggact gtggggcctg
     2221 cgcaaggtgc gggaggagaa cgacagacac cacgccctgg acgccgtggt ggttgcctgc
     2281 agtaccgtga gcatgcagca aaagatcacc gaggccatga agcgcagaga aagcctggag
     2341 acagtgaaca cagagacagg cgaggtgaaa cagagaattc ctcaaccttg ggacttcttc
     2401 agacaggagg tgatgattcg cgtgttcagc gaccacccca gagaggaact cgccgaaaag
     2461 ctgaacagcc ggccacaggc cctgcacgag cacgtggccc ctctcttcgt gtccagagcc
     2521 cctaacagaa agaccaccgg ccagggccac ctggagacca ttaagagcgc caagaggctg
     2581 agcgaaaaca tcagcgtggt gaagaagcct ctggcatctc tcaaaacaaa ggacatcgag
     2641 aacatcgccg gctacaccgc caatagagag cctgagttct accaggctct gaagaagcgc
     2701 cttgccgagt tcaacgacga tcctaaaaag gcttttgccg agcccttcta caagcccacc
     2761 aagaacggcg agcagggccc tctggtgaaa tctgtgagag ttgaggatga ccgggtgaag
     2821 gtgggcctgg ccgtgcggca cggcatcaca gatacaaacg gcgacatggt gcgggccgac
     2881 gtgtaccaga aggccaacaa gtccggcaga atccagtact tcgtggtgcc tgtgtacgcc
     2941 tggcagatcg ccaaaggcgt gctgccactg ctcgactgca gaggctacaa gatcgacgag
     3001 acatatactt tctgcttcag cctgcacaaa tacgacctga tctcattccc taaggacggc
     3061 aacccagagg aagaacagct ggtgtattac ataacctgtg attccagcaa cggcagattc
     3121 tggatcgcca gccacgacaa gggcgctaag gaacagcagt ttagaatctc tacccaaaac
     3181 cagctgttca tcaagaagta ccagaccgac cccctgggca aggagatcag actgtgtaaa
     3241 cctgagcaga gacccgccct gaagatcaag agaaagcggc ccgcagcaac caagaaggca
     3301 ggacaggcca agaagaagaa gtacccctat gacgtgcctg actacgccgg gtatccctac
     3361 gacgtgcctg attacgccgg gtcctatccc tacgacgtgc cagattacgc tgcagctcca
     3421 gcagcgaaga aaaagaagct ggattaagat ctttttccct ctgccaaaaa ttatggggac
     3481 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     3541 atagtgtgtt ggaatttttt gtgtctctca ctcggcggcc gcaggaaccc ctagtgatgg
     3601 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3661 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3721 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3781 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     4141 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4261 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4441 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4501 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4861 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5161 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5341 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6301 ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
     6361 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     6421 gagtggccaa ctccatcact aggggttcct gcggcctcta gagtttaaac aaaaaaataa
     6481 acgatgcccc ttaaagcaga agctttaagg ggcagagcgt tgcggcacat cttttcagac
     6541 ggccttattg tagcaacggt tctcatttcg tttccgaaat gagaaaggga gctacaactg
     6601 aagagctgga atgctcttcc ggtgtttcgt cctttccaca agatatataa agccaagaaa
     6661 tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa
     6721 actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt
     6781 gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg tatatgcaaa
     6841 tatgaaggaa tcatgggaaa taggccctct tcctgcccga ccttgacgtc gactctagaa
     6901 tggaggcggt actatgtaga tgagaattca ggagcaaact gggaaaagca actgcttcca
     6961 aatatttgtg atttttacag tgtagttttg gaaaaactct tagcctacca attcttctaa
     7021 gtgttttaaa atgtgggagc cagtacacat gaagttatag agtgttttaa tgaggcttaa
     7081 atatttaccg taactatgaa atgctacgca tatcatgctg ttcaggctcc gtggccacgc
     7141 aactcatact taagcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa
     7201 caccgccacc atggtg
//
