LOCUS       pAAV_3xgRNA_KO;Template        6659 bp    DNA     circular UNA 11-JUL-2025
DEFINITION  pAAV-U6sgRNA(SapI)_hSyn-GFP-KASH-bGH (SpGuide acceptor). AAV
            plasmid for sgRNA cloning. GFP-KASH fusion facilitates FACS sorting
            of cells and nuclei. .
ACCESSION   urn.local...13-jebled8
VERSION     urn.local...13-jebled8
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     repeat_region   1..141
                     /locus_tag="AAV2 ITR"
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     misc_feature    150..168
                     /standard_name="Primer site #1"
     misc_feature    169..458
                     /standard_name="bU6"
     primer_bind     232..252
                     /standard_name="gBlock_3gRNA_1F"
     misc_feature    459..478
                     /standard_name="gRNA #1"
     misc_feature    461..466
                     /standard_name="BbsI"
     misc_feature    479..564
                     /standard_name="Scaffold cr1"
     misc_feature    565..571
                     /standard_name="polyT"
     misc_feature    572..591
                     /standard_name="Primer site #2"
     misc_feature    592..905
                     /standard_name="mU6"
     protein_bind    786..819
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GCATACAT)."
                     /standard_name="loxP"
     primer_bind     823..843
                     /standard_name="mU6-F"
     primer_bind     complement(884..905)
                     /standard_name="SEQ_mU6_last_R"
     misc_feature    906..925
                     /standard_name="gRNA #2"
     misc_feature    926..1013
                     /standard_name="Scaffold cr2"
     misc_feature    1014..1020
                     /standard_name="polyT"
     misc_feature    1021..1039
                     /standard_name="Primer site #3"
     misc_feature    1068..1316
                     /standard_name="hU6"
     primer_bind     1068..1088
                     /standard_name="hU6-F"
     primer_bind     1239..1258
                     /standard_name="LKO.1 5'"
     misc_feature    1317..1336
                     /standard_name="gRNA #3"
     misc_feature    1329..1334
                     /standard_name="BbsI"
     misc_feature    1337..1424
                     /standard_name="Scaffold cr3"
     misc_feature    1425..1431
                     /standard_name="polyT"
     misc_feature    1432..1453
                     /standard_name="Primer site #4"
     regulatory      1485..1932
                     /note="Geneious type: promoter"
                     /note="human synapsin I promoter; confers neuron-specific
                     expression (<a
                     href=""http://www.ncbi.nlm.nih.gov/pubmed/12595892""
                     title=""http://www.ncbi.nlm.nih.gov/pubmed/12595892"">Kügl
                     er et al., 2003</a>)"
                     /standard_name="hSyn promoter"
     primer_bind     complement(1486..1509)
                     /standard_name="SEQ_hSyn_0R"
     CDS             1962..3030
                     /codon_start=1
                     /locus_tag="smFP HA"
                     /transl_table=1
                     /translation="MYPYDVPDYAGYPYDVPDYAGYPYDVPDYAGGVSKGEELFTGVV
                     PILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLGGGVQC
                     FSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGI
                     DFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEGGYPYDVPDYAGGY
                     PYDVPDYAGGGGYPYDVPDYAGGYPYDVPDYAGGDGSVQLADHYQQNTPIGDGPVLLP
                     DNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGYPYDVPDYAGYPYD
                     VPDYAGYPYDVPDYA*TGARGT"
                     /standard_name="smFP HA"
     CDS             1965..1991
                     /codon_start=1
                     /locus_tag="HA(5)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             1995..2021
                     /codon_start=1
                     /locus_tag="HA(6)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2025..2051
                     /codon_start=1
                     /locus_tag="HA(7)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2580..2606
                     /codon_start=1
                     /locus_tag="HA(8)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2613..2639
                     /codon_start=1
                     /locus_tag="HA"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2652..2678
                     /codon_start=1
                     /locus_tag="HA(1)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2685..2711
                     /codon_start=1
                     /locus_tag="HA(2)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2922..2948
                     /codon_start=1
                     /locus_tag="HA(3)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2952..2978
                     /codon_start=1
                     /locus_tag="HA(4)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     CDS             2982..3008
                     /codon_start=1
                     /locus_tag="HA(9)"
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /transl_table=1
                     /translation="YPYDVPDYA"
                     /standard_name="HA"
     misc_feature    3043..3057
                     /locus_tag="X"
                     /standard_name="X"
     intron          3063
                     /locus_tag="chimeric intron"
                     /note="chimera between introns from human beta-globin and
                     immunoglobulin heavy chain genes"
                     /standard_name="chimeric intron"
     primer_bind     3063..3065
                     /locus_tag="pMT2-F"
                     /note="Synthetic intron, forward primer"
                     /standard_name="pMT2-F"
     misc_feature    3072..3660
                     /locus_tag="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
                     /standard_name="WPRE"
     primer_bind     complement(3125..3145)
                     /locus_tag="WPRE-R"
                     /note="WPRE, reverse primer"
                     /standard_name="WPRE-R"
     CDS             complement(3543..3554)
                     /codon_start=1
                     /locus_tag="Factor Xa site"
                     /product="Factor Xa recognition and cleavage site"
                     /transl_table=1
                     /translation="IEGR"
                     /standard_name="Factor Xa site"
     primer_bind     complement(3663..3680)
                     /locus_tag="BGH-rev"
                     /note="Bovine growth hormone terminator, reverse primer.
                     Also called BGH reverse"
                     /standard_name="BGH-rev"
     misc_feature    3669..3876
                     /note="Geneious type: polyA_signal"
                     /locus_tag="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
                     /standard_name="bGH poly(A) signal"
     misc_feature    3886..3908
                     /locus_tag="DRS1(1)"
                     /standard_name="DRS1"
     repeat_region   3922..4062
                     /locus_tag="AAV2 ITR(2)"
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     rep_origin      4137..4592
     primer_bind     complement(4224..4243)
                     /locus_tag="F1ori-R"
                     /note="F1 origin, reverse primer"
                     /standard_name="F1ori-R"
     primer_bind     4434..4455
                     /locus_tag="F1ori-F"
                     /note="F1 origin, forward primer"
                     /standard_name="F1ori-F"
     primer_bind     complement(4609..4628)
                     /locus_tag="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
                     /standard_name="pRS-marker"
     primer_bind     4728..4750
                     /locus_tag="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
                     /standard_name="pGEX 3'"
     primer_bind     complement(4788..4806)
                     /locus_tag="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
                     /standard_name="pBRforEco"
     regulatory      4874..4978
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /locus_tag="AmpR promoter"
                     /standard_name="AmpR promoter"
     CDS             4979..5839
                     /codon_start=1
                     /gene="bla"
                     /locus_tag="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /product="beta-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
                     /standard_name="AmpR"
     primer_bind     complement(5197..5216)
                     /locus_tag="Amp-R"
                     /note="Ampicillin resistance gene, reverse primer"
                     /standard_name="Amp-R"
     rep_origin      6010..6598
     primer_bind     6499..6518
                     /locus_tag="pBR322ori-F"
                     /note="pBR322 origin, forward primer"
                     /standard_name="pBR322ori-F"
ORIGIN      
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccggt aggtccctcg acgttaaccg tgaccgagct
      181 tgtctgcccg cgcagtccac tagacagacg cgcagaggtc gggcgggcct agggccaatt
      241 tcccatggtt cccttcattt gcatatatga tgtaataagg ttatggagac tattaaactt
      301 agccctaatc aaactatatg atgataatgt gtgtggtaca aaaggtcata acttattata
      361 tactttgaaa cttaaaaaag ggttacagtt tagtcaccat aactgtaaaa ttttttctat
      421 tcttagcttt atatagttct tgagaggcca tgtttatgct gtcttcatca tggccgacgt
      481 ttaagagcta agctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt
      541 gaaaaagtgg caccgagtcg gtgctttttt tccagtctga ctatcacgat ggatccgacg
      601 cgccatctct aggcccgcgc cggccccctc gcacggactt gtgggagaag ctcggctact
      661 cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc ttaatgtgcg
      721 ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga taggcttgga
      781 tttctataac ttcgtatagc atacattata cgaagttata aacagcacaa aaggaaactc
      841 accctaactg taaagtaatt gtgtgttttg agactataag tatcccttgg agaaccacct
      901 tgttggttcg cattatccga accatgtttg agagctaagc agaaagctgc atagcaagtt
      961 caaataaggc tagtccgtac acaacttgaa aaagtggcag ccgagtcggc tgcttttttt
     1021 gctcgaatct acactcagct atggcgcgcc ccaaggtcgg gcaggaagag ggcctatttc
     1081 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     1141 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     1201 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     1261 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaagccaga aacatgtaag
     1321 cgtcgcaaga agacgggttt cagagctaag cacaagagtg catagcaagt tgaaataagg
     1381 ctagtccgtt tacaacttga aaaagtggca cccgagtcgg gtgctttttt tctcgagtac
     1441 taggatccat taggcggtct agactgcaga gggccctgcg tatgagtgca agtgggtttt
     1501 aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga cccactggac
     1561 aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg gaggggaaac
     1621 aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt gccttcgccc
     1681 ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc
     1741 ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc cgccggccca
     1801 gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc
     1861 ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc
     1921 ctgagagcgc agtcgagaag gtaccggatc cggccgccac catgtaccct tatgatgtgc
     1981 ccgattatgc tggctaccct tatgatgttc ctgattacgc cggatatccg tatgacgtgc
     2041 cagactacgc gggaggtgtg agcaagggag aggagttgtt taccggcgtg gtccccatac
     2101 tggtggagtt ggatggcgac gttaatggac ataaattctc ggtccgcggc gagggagagg
     2161 gagacgccac caatggcaag ctgacgctta agttcatttg cacgactgga aaattgcctg
     2221 tcccctggcc gaccctggtc accacactgg gcggaggagt gcagtgcttc tcccgttacc
     2281 cagaccacat gaagcagcac gatttcttca agagcgcaat gccggaggga tacgtgcaag
     2341 aacggactat ttccttcaag gatgacggaa cctataagac ccgtgcggag gtcaaattcg
     2401 agggtgacac cctggtgaac cgaattgaac tcaaaggaat cgatttcaag gaggatggaa
     2461 atattctggg tcacaagctg gaatacaact tcaacagcca taatgtgtac attacggctg
     2521 ataagcagaa gaacggcatt aaggccaatt tcaagatccg ccacaacgtt gagggtggat
     2581 atccctacga cgtgcccgat tatgcgggcg gatatccgta tgatgttcca gattatgctg
     2641 gtggtggagg ctatccctat gatgtccccg actacgccgg aggttaccca tacgacgtgc
     2701 ccgactacgc tggaggcgat ggcagcgtgc agctggcaga tcattatcaa cagaataccc
     2761 ccataggtga tggccccgtt ctgcttccag ataatcacta cctttccacc cagagcgtgc
     2821 tttcgaaaga cccgaacgaa aagcgtgatc acatggtcct gctggagttt gtgaccgcgg
     2881 ctggaatcac cctgggtatg gacgaactct acaagggtgg ctacccctac gatgtgccgg
     2941 attacgctgg atatccgtat gacgtaccgg actatgccgg ttacccgtac gatgtcccgg
     3001 actacgctta aaccggtgct cgaggtaccg aattcgatat catagctagc tagctaggga
     3061 tcggtgtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3121 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3181 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3241 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3301 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3361 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3421 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     3481 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3541 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3601 ttccgcgtct acgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3661 ctcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3721 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3781 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3841 ggaggattgg gaagagaata gcaggcatgc tggggagcgg gatccgacgc ttccgagtac
     3901 ggtacaggct agaggggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3961 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4021 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4081 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4201 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4321 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4441 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     4501 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4561 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4621 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4681 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4741 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4801 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4861 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4921 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4981 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5041 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5101 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5161 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5221 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5281 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5341 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5401 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5461 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5521 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5581 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5641 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5701 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5761 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5821 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5881 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5941 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6001 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6061 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6121 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
