LOCUS       sequence-176238-        3020 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Express sgRNA targeting human AKT3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3020)
  AUTHORS   Hazelbaker DZ, Beccard A, Bara AM, Dabkowski N, Messana A, Mazzucato
            P, Lam D, Manning D, Eggan K, Barrett LE
  TITLE     A Scaled Framework for CRISPR Editing of Human Pluripotent Stem 
            Cells to Study Psychiatric Disease.
  JOURNAL   Stem Cell Reports. 2017 Oct 10;9(4):1315-1327. doi: 
            10.1016/j.stemcr.2017.09.006.
  PUBMED    29020615
REFERENCE   2  (bases 1 to 3020)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stemcr.2017.09"; journalName: "Stem Cell Reports"; date: 
            "2017-10-10- 10"; volume: "9"; issue: "4"; pages: "1315-1327"
COMMENT     Sequence Label: pPN186
FEATURES             Location/Qualifiers
     source          1..3020
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        432..680
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     432..452
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     603..622
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        699..774
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(799..815)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(799..815)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(812..834)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    823..839
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(847..853,854..871,872..877))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    892..913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1030..1047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1201..1789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1281..1300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1960..2751,2752..2820))
                     /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     2583..2602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2821..2925)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2993..3011
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      421 ccgttaatta agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      481 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      541 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      601 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      661 tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa
      721 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      781 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      841 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      901 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      961 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1021 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1081 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1141 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1201 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1261 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1321 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1381 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1441 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1501 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1561 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1621 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1681 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1741 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1801 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1861 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1921 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1981 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2041 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2101 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2161 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2221 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2281 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2341 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2401 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2461 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2521 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2581 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2641 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2701 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2761 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2821 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2941 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3001 tatcacgagg ccctttcgtc
//
