LOCUS       Exported                5292 bp ds-DNA     circular SYN 25-JAN-2023
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   .
VERSION     .
KEYWORDS    PX552
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5292)
  AUTHORS   Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur
            M, Zhang F
  TITLE     In vivo interrogation of gene function in the mammalian brain using 
            CRISPR-Cas9.
  JOURNAL   Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055.
  PUBMED    25326897
REFERENCE   2  (bases 1 to 5292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 25, 2023 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5292
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..385
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        521..968
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1000..1009
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1006..1722
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1006..1722
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2039..2515
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2238..2257)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2555..2695
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2555..2684
                     /label=AAV2 ITR
     rep_origin      2770..3225
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2857..2876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3067..3088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3242..3261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3361..3383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3421..3439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3507..3611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3612..4472
                     /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(3830..3849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4643..5231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5132..5151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt ttttctagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
      541 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
      601 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
      661 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
      721 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
      781 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
      841 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
      901 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
      961 gagcgcagtc gagaaggtac cggatcctct agagtcgacg ccaccatggt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1201 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact cagatctcga
     1741 gaggaggagg aggagacaga cagcaggatg ccccacctcg acagccccgg cagctcccag
     1801 ccgagacgct ccttcctctc aagggtgatc agggcagcgc taccgttgca gctgcttctg
     1861 ctgctgctgc tgctcctggc ctgcctgcta cctgcctctg aagatgacta cagctgcacc
     1921 caggccaaca actttgcccg atccttctac cccatgctgc ggtacaccaa cgggccacct
     1981 cccacctagg aattcgatat caagcttatc gataccgagc gctgctcgag agatctacgg
     2041 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     2101 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     2161 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     2221 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     2281 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     2341 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     2401 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     2461 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     2521 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     2581 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     2641 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     2701 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     2761 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2821 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     2881 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2941 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     3001 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3061 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     3121 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     3181 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     3241 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     3301 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3361 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     3421 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3481 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3601 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3661 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3721 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3781 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3841 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3901 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3961 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4021 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4081 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4141 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4201 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4261 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4321 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4381 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4441 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4501 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4561 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4621 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4681 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4741 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4801 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4861 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4921 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4981 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5041 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5101 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5161 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5221 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5281 ttgctcacat gt
//
