LOCUS       Exported                6881 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  SaCas9 driven by EFS. SaCas9 is followed by the self cleaving P2A 
            sequence, several tags, and then the KASH transmembrane domain to 
            enable FACS..
ACCESSION   .
VERSION     .
KEYWORDS    pJEP311-pAAV-EFS-SaCas9-P2A-HAFLAGHA-KASH-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6881)
  AUTHORS   Kumar N, Stanford W, Aradhana CS, Abraham ND, Dao TJ, Thaseen S, 
            Sairavi A, Gonzalez CU, Ploski JE.
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front. Mol. Neurosci. (2018) 13
REFERENCE   2  (bases 1 to 6881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6881
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        115..326
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      347..356
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             359..379
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             404..3559
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3560..3607
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3623..3679
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3683..3709
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3710..3733
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3734..3760
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4034..4082
                     /note="synthetic polyadenylation signal"
     repeat_region   4090..4230
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4090..4219
                     /label=AAV2 ITR
     rep_origin      4305..4760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4392..4411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4602..4623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4777..4796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4896..4918
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4956..4974)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5042..5146
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5147..6007
                     /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(5365..5384)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6178..6766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6667..6686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6828..76
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
       61 atcactaggg gttcctgcgg cctctagagt cgagtggctc cggtgcccgt cagtgggcag
      121 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaacgggtg
      181 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      241 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      301 gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg accggtgcca ccatggcccc
      361 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccaagcgga actacatcct
      421 gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga
      481 cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgagggcag
      541 gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa tccagagagt
      601 gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa
      661 cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc
      721 cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga
      781 caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctggaaga
      841 gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag
      901 catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca
      961 gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc tgctggaaac
     1021 ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa
     1081 agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac tgcggagcgt
     1141 gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat
     1201 caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt
     1261 gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga
     1321 agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca ccaacctgaa
     1381 ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct
     1441 gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga
     1501 actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct ctaatctgaa
     1561 gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc tggacgagct
     1621 gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa
     1681 ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag
     1741 ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa
     1801 gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc
     1861 ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc ggatcgagga
     1921 aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga tcaagctgca
     1981 cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct
     2041 gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa
     2101 cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac
     2161 cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca
     2221 catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct
     2281 ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt
     2341 ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact tcagagtgaa
     2401 caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc tgcggcggaa
     2461 gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat
     2521 cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt
     2581 gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga
     2641 gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta aggacttcaa
     2701 ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac
     2761 cctgtactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg
     2821 cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct
     2881 gctgatgtac caccacgacc cccagaccta ccagaaactg aagctgatta tggaacagta
     2941 cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa
     3001 gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact
     3061 gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct
     3121 gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac
     3181 cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta
     3241 tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg cctccttcta
     3301 caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga
     3361 cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt acctggaaaa
     3421 catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga cccagagcat
     3481 taagaagtac agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc
     3541 tcagatcatc aaaaagggca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     3601 gaaaaaggga tccggaagcg gagctactaa cttcagcctg ctgaagcagg ctggcgacgt
     3661 ggaggagaac cctggacctg gttacccata cgatgttcca gattacgctg actacaaaga
     3721 cgatgacgac aagtacccat acgatgttcc agattacgct tccggactca gatctcgaga
     3781 ggaggaggag gagacagaca gcaggatgcc ccacctcgac agccccggca gctcccagcc
     3841 gagacgctcc ttcctctcaa gggtgatcag ggcagcgcta ccgttgcagc tgcttctgct
     3901 gctgctgctg ctcctggcct gcctgctacc tgcctctgaa gatgactaca gctgcaccca
     3961 ggccaacaac tttgcccgat ccttctaccc catgctgcgg tacaccaacg ggccacctcc
     4021 cacctaggaa ttcaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg
     4081 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4141 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4201 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4261 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4321 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4381 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4441 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4501 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4561 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4621 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4681 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4741 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4801 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4861 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4921 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4981 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5041 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5101 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5161 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5221 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5281 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5341 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5401 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5461 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5521 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5581 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5641 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5701 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5761 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5821 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     5881 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5941 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6001 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6061 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6121 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6181 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6241 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6301 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6361 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6421 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6481 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6541 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6601 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6661 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6721 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6781 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct
     6841 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc g
//
