LOCUS       Exported                7214 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  SaCas9 driven by CMV. SaCas9 is followed by the self cleaving P2A 
            sequence, several tags, and then the KASH transmembrane domain to 
            enable FACS.
ACCESSION   .
VERSION     .
KEYWORDS    pJEP312-pAAV-CMV-SaCas9-P2A-HAFLAGHA-KASH-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7214)
  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 7214)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7214
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      532..1120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1021..1040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1182..1311
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        1333..1636
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1637..1840
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1791..1811
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1837..1861
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1915..1924
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1927..1947
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1972..5127
                     /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             5128..5175
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5191..5247
                     /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             5251..5277
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5278..5301
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5302..5328
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5602..5650
                     /note="synthetic polyadenylation signal"
     repeat_region   5658..5798
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5658..5787
                     /label=AAV2 ITR
     rep_origin      5873..6328
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5960..5979)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6170..6191
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6345..6364)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6464..6486
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6524..6542)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6610..6714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6715..361
                     /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(6933..6952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
       61 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      181 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
      241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      661 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1141 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
     1201 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     1261 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggcctct
     1321 agaggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1381 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     1441 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     1501 gtatcatatg ccaagtacgc cccctattga cgtcaatgac agtaaatggc ccgcctggca
     1561 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     1621 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     1681 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     1741 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     1801 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
     1861 cgcctggaga cgccatccac gctgttttga cctccataga agacaccgac cggtgccacc
     1921 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc caagcggaac
     1981 tacatcctgg gcctggacat cggcatcacc agcgtgggct acggcatcat cgactacgag
     2041 acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac
     2101 gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg gcatagaatc
     2161 cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag cgagctgagc
     2221 ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag cgaggaagag
     2281 ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt gaacgaggtg
     2341 gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa cagcaaggcc
     2401 ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg
     2461 cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa acagctgctg
     2521 aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta catcgacctg
     2581 ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt cggctggaag
     2641 gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc cgaggaactg
     2701 cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga cctgaacaat
     2761 ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc
     2821 gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc
     2881 gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc
     2941 aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac
     3001 gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc
     3061 caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct
     3121 aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg
     3181 gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg
     3241 cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc
     3301 atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc
     3361 atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc
     3421 aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg
     3481 atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc
     3541 aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa
     3601 gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc
     3661 ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc
     3721 aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc
     3781 aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag
     3841 tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg
     3901 aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc
     3961 agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg
     4021 cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac
     4081 gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc
     4141 aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc
     4201 gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag
     4261 gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt
     4321 aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat
     4381 ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc
     4441 gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg
     4501 gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac
     4561 ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc
     4621 aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc
     4681 gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag
     4741 ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc
     4801 aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc
     4861 tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg
     4921 aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac
     4981 ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc
     5041 cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag
     5101 aagcaccctc agatcatcaa aaagggcaaa aggccggcgg ccacgaaaaa ggccggccag
     5161 gcaaaaaaga aaaagggatc cggaagcgga gctactaact tcagcctgct gaagcaggct
     5221 ggcgacgtgg aggagaaccc tggacctggt tacccatacg atgttccaga ttacgctgac
     5281 tacaaagacg atgacgacaa gtacccatac gatgttccag attacgcttc cggactcaga
     5341 tctcgagagg aggaggagga gacagacagc aggatgcccc acctcgacag ccccggcagc
     5401 tcccagccga gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg
     5461 cttctgctgc tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc
     5521 tgcacccagg ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg
     5581 ccacctccca cctaggaatt caataaaaga tctttatttt cattagatct gtgtgttggt
     5641 tttttgtgtg cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     5701 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     5761 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5821 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5881 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5941 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6001 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6061 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     6121 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6181 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     6241 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6301 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     6361 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6421 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6481 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6541 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     7021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     7081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     7141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     7201 tgggaaccgg agct
//
