LOCUS       Exported                7289 bp ds-DNA     circular SYN 16-MAY-2021
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7289)
  AUTHORS   Subhojit Roy
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Apr 29, 2025 from SnapGene 5.1.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7289
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
                     /note="color: #ffcc99"
     promoter        143..590
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      597..606
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             609..629
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             654..3809
                     /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"
                     /note="color: #993366"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3810..3857
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3864..3944
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /note="color: #cc99b2"
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    3978..4185
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     promoter        4192..4432
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    4442..4462
                     /label=Scramble control
                     /note="color: #ffff00"
     misc_RNA        4463..4538
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     repeat_region   4552..4692
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     repeat_region   4552..4681
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     rep_origin      4767..5222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        5504..5608
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5609..6469
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5609 .. 5677 / #ccffcc / signal sequence
                        2: 5678 .. 6469 / #ccffcc
                     Cleavage site after base 5677"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6640..7228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaagtgcaag tgggttttag gaccaggatg aggcggggtg
      181 ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc
      241 aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg
      301 cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc
      361 gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc
      421 ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc
      481 gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc
      541 ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag accggtgcca
      601 ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccaagcgga
      661 actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg
      721 agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca
      781 acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa
      841 tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga
      901 gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag
      961 agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg
     1021 tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg
     1081 ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag
     1141 tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc
     1201 tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc
     1261 tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga
     1321 aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac
     1381 tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca
     1441 atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca
     1501 tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc
     1561 tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca
     1621 ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga
     1681 acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca
     1741 tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct
     1801 ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc
     1861 tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg
     1921 tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact
     1981 tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca
     2041 tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact
     2101 ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc
     2161 ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga
     2221 tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg
     2281 aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt
     2341 ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg
     2401 gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct
     2461 tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag
     2521 agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc
     2581 ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact
     2641 tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc
     2701 tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg
     2761 acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg
     2821 ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga
     2881 tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta
     2941 aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga
     3001 ttaacgacac cctgtactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca
     3061 atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc
     3121 ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg aagctgatta
     3181 tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact
     3241 acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg
     3301 gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg
     3361 tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca
     3421 agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata
     3481 gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg
     3541 cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg
     3601 tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt
     3661 acctggaaaa catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga
     3721 cccagagcat taagaagtac agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta
     3781 agaagcaccc tcagatcatc aaaaagggca aaaggccggc ggccacgaaa aaggccggcc
     3841 aggcaaaaaa gaaaaaggga tcctacccat acgatgttcc agattacgct tacccatacg
     3901 atgttccaga ttacgcttac ccatacgatg ttccagatta cgcttaagaa ttcctagagc
     3961 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4021 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4081 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4141 cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac cgagggccta
     4201 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     4261 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     4321 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     4381 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
     4441 gacggaggct aagcgtcgca aggttttagt actctggaaa cagaatctac taaaacaagg
     4501 caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttgcggccg caggaacccc
     4561 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4621 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4681 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4741 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4801 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4861 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4921 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4981 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5041 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5101 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5161 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5221 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5281 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5341 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5401 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5461 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5521 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5581 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5641 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5701 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5761 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5821 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5881 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5941 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6001 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6061 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6121 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6181 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6241 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6301 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     6361 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6421 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6481 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6541 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6601 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6661 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6721 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6781 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6841 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6901 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6961 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7021 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7081 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7141 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7201 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7261 ggccttttgc tggccttttg ctcacatgt
//
