LOCUS       2025_AAV_H1-GRIN        6508 bp DNA     circular SYN 02-MAR-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6508)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Sep 27, 2024 from SnapGene Viewer 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     misc_feature    1..130
                     /label=L-ITR
     primer_bind     133..170
                     /label=Primer 2
     misc_feature    137..142
                     /label=XbaI
                     /note="XbaI
                     Geneious type: restriction"
     misc_feature    146..242
                     /label=H1 promoter with additional G
     misc_RNA        269..344
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    272
                     /label=G/C/A?
     misc_feature    299
                     /label=?C/G/T
     CDS             311..364
                     /codon_start=1
                     /label=Feature 54
                     /translation="MPCLSRQLVGEIFSTGAT"
     primer_bind     complement(318..362)
                     /label=Primer 5
     primer_bind     complement(324..349)
                     /label=SaCas_Scaffold_ClaI_re
     misc_feature    359..368
                     /label=Kozak
     primer_bind     364..388
                     /label=#609_SaCas9_BbsI_v2_fw
     misc_feature    365..368
                     /label=NLS
                     /note="NLS"
     misc_feature    369..415
                     /label=NLS
                     /note="NLS"
     primer_bind     392..418
                     /label=SaCas9_BbsI_fw
     CDS             416..3571
                     /codon_start=1
                     /product="Cas9 (Csn1) 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"
     misc_feature    815..817
                     /label=Ub high
                     /note="Ub high"
     primer_bind     909..931
                     /label=hSaCas9_Seq1_fw
     primer_bind     complement(1694..1714)
                     /label=MH_SaN_Split_434_BbsI_BsmBI_re
     primer_bind     complement(1696..1718)
                     /label=MH_SaN_Split_435_BbsI_BsmBI_re
     primer_bind     complement(1697..1720)
                     /label=MH_SaN_Split_436_BbsI_BsmBI_re
     primer_bind     complement(1702..1724)
                     /label=MH_SaN_Split_437_BbsI_BsmBI_re
     primer_bind     complement(1705..1727)
                     /label=MH_SaN_Split_438_BbsI_BsmBI_re
     primer_bind     1715..1733
                     /label=MH_SaC_Split_435_BbsI_BsmBI_fw
     primer_bind     1718..1735
                     /label=MH_SaC_Split_436_BbsI_BsmBI_fw
     primer_bind     1721..1738
                     /label=MH_SaC_Split_437_BbsI_BsmBI_fw
     primer_bind     1724..1741
                     /label=MH_SaC_Split_438_BbsI_BsmBI_fw
     primer_bind     1725..1744
                     /label=hSaCas9_Seq2_fw
     primer_bind     1727..1747
                     /label=MH_SaC_Split_439_BbsI_BsmBI_fw
     primer_bind     2538..2561
                     /label=hSaCas9_Seq3_fw
     primer_bind     complement(2569..2594)
                     /label=SaN_split_727_BbsI_BsmBI_re
     primer_bind     complement(2591..2614)
                     /label=DN_SaN_split734_BbsI_BsmBI_re
     primer_bind     2594..2620
                     /label=SaC_split_728_BbsI_BsmBI_fw
     CDS             2600..2647
                     /codon_start=1
                     /label=split loop 2
                     /translation="NQMFEEKQAESMPEIE"
     primer_bind     complement(2608..2631)
                     /label=DN_SaN_split739_BbsI_BsmBI_re
     primer_bind     complement(2608..2631)
                     /label=SaCasN_split739_BbsI_BsmBI_re
     primer_bind     complement(2608..2629)
                     /label=SaCas9_739_BbsI_re
     CDS             2612..2617
                     /codon_start=1
                     /label=Aplit 734/735
                     /translation="EE"
     primer_bind     complement(2615..2642)
                     /label=DN_SaN_split743_BbsI_BsmBI_re
     primer_bind     2615..2636
                     /label=DN_SaC_split735_BbsI_BsmBI_fw
     misc_feature    2618..2620
                     /label=Ub high - AAG = K (original), AGG = R
                     /note="Ub high - AAG = K (original), AGG = R"
     primer_bind     complement(2627..2648)
                     /label=SaN_split_745_BbsI_BsmBI_re
     CDS             2627..2632
                     /codon_start=1
                     /label=split 739/740
                     /translation="ES"
     primer_bind     2630..2650
                     /label=DN_SaC_split740_BbsI_BsmBI_fw
     CDS             2639..2644
                     /codon_start=1
                     /label=Split 743/744
                     /translation="EI"
     primer_bind     2642..2666
                     /label=DN_SaC_split744_BbsI_BsmBI_fw
     primer_bind     2648..2671
                     /label=SaC_split_746_BbsI_BsmBI_fw
     primer_bind     3355..3374
                     /label=hSaCas9_Seq4_fw
     primer_bind     complement(3547..3573)
                     /label=SaCas9_BbsI_re
     misc_feature    3572..3619
                     /label=NLS
                     /note="NLS"
     misc_feature    3620
                     /label=BamHI
                     /note="BamHI"
     misc_feature    3708..3767
                     /label=polyA
     misc_feature    3771..3911
                     /label=R-ITR
     rep_origin      4003..4309
                     /label=f1 Origin
     misc_feature    4828..5685
                     /label=AmpR
     rep_origin      5836..6503
                     /label=pUC ori
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactccatat ttgcatgtcg ctatgtgttc tgggaaatca
      181 ccataaacgt gaaatgtctt tggatttggg aatcttataa gttctgtatg aggaccacgg
      241 tacaccgaga gtaggctggt agatggaggt tttagtactc tggaaacaga atctactaaa
      301 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt ccaccggtgc
      361 caccatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccaagcg
      421 gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca tcatcgacta
      481 cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca acgtggaaaa
      541 caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga ggcggcatag
      601 aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc acagcgagct
      661 gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc tgagcgagga
      721 agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca acgtgaacga
      781 ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc ggaacagcaa
      841 ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga aagacggcga
      901 agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag ccaaacagct
      961 gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca cctacatcga
     1021 cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc ccttcggctg
     1081 gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact tccccgagga
     1141 actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga acgacctgaa
     1201 caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga agttccagat
     1261 catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg ccaaagaaat
     1321 cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca agcccgagtt
     1381 caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag agattattga
     1441 gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga gcagcgagga
     1501 catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga tcgagcagat
     1561 ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca tcaacctgat
     1621 cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc ggctgaagct
     1681 ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc tggtggacga
     1741 cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag tgatcaacgc
     1801 catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc gcgagaagaa
     1861 ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaaccggc agaccaacga
     1921 gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc tgatcgagaa
     1981 gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag ccatccctct
     2041 ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc ccagaagcgt
     2101 gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaaa acagcaagaa
     2161 gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca gctacgaaac
     2221 cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca agaccaagaa
     2281 agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag acttcatcaa
     2341 ccggaacctg gtggatacca gatacgccac cagaggcctg atgaacctgc tgcggagcta
     2401 cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct tcaccagctt
     2461 tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc accacgccga
     2521 ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga aactggacaa
     2581 ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag caggccgaga gcatgcccga
     2641 gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga tcaagcacat
     2701 taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta atagagagct
     2761 gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc tgatcgtgaa
     2821 caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga tcaacaagag
     2881 ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac tgaagctgat
     2941 tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg aaaccgggaa
     3001 ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga ttaagtatta
     3061 cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca gcagaaacaa
     3121 ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca atggcgtgta
     3181 caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact acgaagtgaa
     3241 tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg ccgagtttat
     3301 cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata gagtgatcgg
     3361 cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca cctaccgcga
     3421 gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa tcgcctccaa
     3481 gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg aagtgaaatc
     3541 taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga aaaaggccgg
     3601 ccaggcaaaa aagaaaaagg gatccgctac aaactttagc cttctcaaac aagcaggaga
     3661 tgtcgaagag aatcctggcc cggtcttctt ttgaagactg agaattctag caataaagga
     3721 tcgtttattt tcattggaag cgtgtgttgg ttttttgatc aggcgcgggc aggaacccct
     3781 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3841 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     3901 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3961 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4021 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4081 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4141 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4201 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4261 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4321 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4381 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     4441 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4501 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4561 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4621 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4681 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4741 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4801 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4861 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4921 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4981 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5041 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5101 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5161 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5221 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5281 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5341 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5401 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5461 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5521 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     5581 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6001 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6121 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6481 gccttttgct ggccttttgc tcacatgt
//
