LOCUS       Exported                6864 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  SaCas9 driven by the neuron specific promoter Mecp2P. SaCas9 is 
            followed by the self cleaving P2A sequence, several tags, and then 
            the KASH transmembrane domain to enable FACS..
ACCESSION   .
VERSION     .
KEYWORDS    pJEP310-pAAV-Mecp2P-SaCas9-P2A-HAFLAGHA-KASH-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6864)
  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 6864)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6864
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     56..75
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   217..346
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        365..593
                     /label=Mecp2 promoter
                     /note="promoter for selective expression in neurons (Adachi
                     et al., 2005; Gray et al., 2011)"
     regulatory      600..609
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             612..632
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             657..3812
                     /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             3813..3860
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3876..3932
                     /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             3936..3962
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3963..3986
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3987..4013
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4287..4335
                     /note="synthetic polyadenylation signal"
     repeat_region   4343..4483
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4343..4472
                     /label=AAV2 ITR
     rep_origin      4558..5013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4645..4664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4855..4876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5030..5049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5149..5171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5209..5227)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5295..5399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5400..6260
                     /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(5618..5637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6431..155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
       61 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      121 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      181 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
      241 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
      301 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagaaa
      361 gcttagctga atggggtccg cctcttttcc ctgcctaaac agacaggaac tcctgccaat
      421 tgagggcgtc accgctaagg ctccgcccca gcctgggctc cacaaccaat gaagggtaat
      481 ctcgacaaag agcaaggggt ggggcgcggg cgcgcaggtg cagcagcaca caggctggtc
      541 gggagggcgg ggcgcgacgt ctgccgtgcg gggtcccggc atcggttgcg cgcaccggtg
      601 ccaccatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccaagc
      661 ggaactacat cctgggcctg gacatcggca tcaccagcgt gggctacggc atcatcgact
      721 acgagacacg ggacgtgatc gatgccggcg tgcggctgtt caaagaggcc aacgtggaaa
      781 acaacgaggg caggcggagc aagagaggcg ccagaaggct gaagcggcgg aggcggcata
      841 gaatccagag agtgaagaag ctgctgttcg actacaacct gctgaccgac cacagcgagc
      901 tgagcggcat caacccctac gaggccagag tgaagggcct gagccagaag ctgagcgagg
      961 aagagttctc tgccgccctg ctgcacctgg ccaagagaag aggcgtgcac aacgtgaacg
     1021 aggtggaaga ggacaccggc aacgagctgt ccaccaaaga gcagatcagc cggaacagca
     1081 aggccctgga agagaaatac gtggccgaac tgcagctgga acggctgaag aaagacggcg
     1141 aagtgcgggg cagcatcaac agattcaaga ccagcgacta cgtgaaagaa gccaaacagc
     1201 tgctgaaggt gcagaaggcc taccaccagc tggaccagag cttcatcgac acctacatcg
     1261 acctgctgga aacccggcgg acctactatg agggacctgg cgagggcagc cccttcggct
     1321 ggaaggacat caaagaatgg tacgagatgc tgatgggcca ctgcacctac ttccccgagg
     1381 aactgcggag cgtgaagtac gcctacaacg ccgacctgta caacgccctg aacgacctga
     1441 acaatctcgt gatcaccagg gacgagaacg agaagctgga atattacgag aagttccaga
     1501 tcatcgagaa cgtgttcaag cagaagaaga agcccaccct gaagcagatc gccaaagaaa
     1561 tcctcgtgaa cgaagaggat attaagggct acagagtgac cagcaccggc aagcccgagt
     1621 tcaccaacct gaaggtgtac cacgacatca aggacattac cgcccggaaa gagattattg
     1681 agaacgccga gctgctggat cagattgcca agatcctgac catctaccag agcagcgagg
     1741 acatccagga agaactgacc aatctgaact ccgagctgac ccaggaagag atcgagcaga
     1801 tctctaatct gaagggctat accggcaccc acaacctgag cctgaaggcc atcaacctga
     1861 tcctggacga gctgtggcac accaacgaca accagatcgc tatcttcaac cggctgaagc
     1921 tggtgcccaa gaaggtggac ctgtcccagc agaaagagat ccccaccacc ctggtggacg
     1981 acttcatcct gagccccgtc gtgaagagaa gcttcatcca gagcatcaaa gtgatcaacg
     2041 ccatcatcaa gaagtacggc ctgcccaacg acatcattat cgagctggcc cgcgagaaga
     2101 actccaagga cgcccagaaa atgatcaacg agatgcagaa gcggaaccgg cagaccaacg
     2161 agcggatcga ggaaatcatc cggaccaccg gcaaagagaa cgccaagtac ctgatcgaga
     2221 agatcaagct gcacgacatg caggaaggca agtgcctgta cagcctggaa gccatccctc
     2281 tggaagatct gctgaacaac cccttcaact atgaggtgga ccacatcatc cccagaagcg
     2341 tgtccttcga caacagcttc aacaacaagg tgctcgtgaa gcaggaagaa aacagcaaga
     2401 agggcaaccg gaccccattc cagtacctga gcagcagcga cagcaagatc agctacgaaa
     2461 ccttcaagaa gcacatcctg aatctggcca agggcaaggg cagaatcagc aagaccaaga
     2521 aagagtatct gctggaagaa cgggacatca acaggttctc cgtgcagaaa gacttcatca
     2581 accggaacct ggtggatacc agatacgcca ccagaggcct gatgaacctg ctgcggagct
     2641 acttcagagt gaacaacctg gacgtgaaag tgaagtccat caatggcggc ttcaccagct
     2701 ttctgcggcg gaagtggaag tttaagaaag agcggaacaa ggggtacaag caccacgccg
     2761 aggacgccct gatcattgcc aacgccgatt tcatcttcaa agagtggaag aaactggaca
     2821 aggccaaaaa agtgatggaa aaccagatgt tcgaggaaaa gcaggccgag agcatgcccg
     2881 agatcgaaac cgagcaggag tacaaagaga tcttcatcac cccccaccag atcaagcaca
     2941 ttaaggactt caaggactac aagtacagcc accgggtgga caagaagcct aatagagagc
     3001 tgattaacga caccctgtac tccacccgga aggacgacaa gggcaacacc ctgatcgtga
     3061 acaatctgaa cggcctgtac gacaaggaca atgacaagct gaaaaagctg atcaacaaga
     3121 gccccgaaaa gctgctgatg taccaccacg acccccagac ctaccagaaa ctgaagctga
     3181 ttatggaaca gtacggcgac gagaagaatc ccctgtacaa gtactacgag gaaaccggga
     3241 actacctgac caagtactcc aaaaaggaca acggccccgt gatcaagaag attaagtatt
     3301 acggcaacaa actgaacgcc catctggaca tcaccgacga ctaccccaac agcagaaaca
     3361 aggtcgtgaa gctgtccctg aagccctaca gattcgacgt gtacctggac aatggcgtgt
     3421 acaagttcgt gaccgtgaag aatctggatg tgatcaaaaa agaaaactac tacgaagtga
     3481 atagcaagtg ctatgaggaa gctaagaagc tgaagaagat cagcaaccag gccgagttta
     3541 tcgcctcctt ctacaacaac gatctgatca agatcaacgg cgagctgtat agagtgatcg
     3601 gcgtgaacaa cgacctgctg aaccggatcg aagtgaacat gatcgacatc acctaccgcg
     3661 agtacctgga aaacatgaac gacaagaggc cccccaggat cattaagaca atcgcctcca
     3721 agacccagag cattaagaag tacagcacag acattctggg caacctgtat gaagtgaaat
     3781 ctaagaagca ccctcagatc atcaaaaagg gcaaaaggcc ggcggccacg aaaaaggccg
     3841 gccaggcaaa aaagaaaaag ggatccggaa gcggagctac taacttcagc ctgctgaagc
     3901 aggctggcga cgtggaggag aaccctggac ctggttaccc atacgatgtt ccagattacg
     3961 ctgactacaa agacgatgac gacaagtacc catacgatgt tccagattac gcttccggac
     4021 tcagatctcg agaggaggag gaggagacag acagcaggat gccccacctc gacagccccg
     4081 gcagctccca gccgagacgc tccttcctct caagggtgat cagggcagcg ctaccgttgc
     4141 agctgcttct gctgctgctg ctgctcctgg cctgcctgct acctgcctct gaagatgact
     4201 acagctgcac ccaggccaac aactttgccc gatccttcta ccccatgctg cggtacacca
     4261 acgggccacc tcccacctag gaattcaata aaagatcttt attttcatta gatctgtgtg
     4321 ttggtttttt gtgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4381 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4441 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4501 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4561 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4621 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4681 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4741 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4801 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4861 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4921 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4981 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     5041 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5101 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5161 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5221 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5281 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5401 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5461 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5521 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5581 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5641 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5701 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5761 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5821 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5881 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5941 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6001 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6061 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     6121 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6181 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6241 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6301 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6361 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6421 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6481 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6541 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6601 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6661 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6721 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6781 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6841 ttcccgaagg gagaaaggcg gaca
//
