LOCUS       sequence-180143-        3023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express sgRNA targeting human CNOT1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3023)
  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 3023)
  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: pPN254
FEATURES             Location/Qualifiers
     source          1..3023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(371..387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(371..387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(384..406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    395..411
                     /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(419..425,426..443,444..449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    464..485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(602..619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(773..1361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(853..872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1532..2323,2324..2392))
                     /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     2155..2174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2393..2497)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2565..2583
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2621..2643)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2743..2762
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2956..2978
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2970..2987
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2971..2987
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg aataaatgcc tgtctgcctc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttaagcttgg
      361 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
      421 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
      481 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
      601 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
      661 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      721 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      781 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      841 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      901 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      961 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1021 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1081 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1141 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1201 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1261 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1321 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1381 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1441 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1501 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1561 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1621 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1681 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1741 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1801 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1861 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1921 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1981 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2041 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2101 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2161 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2221 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2281 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2341 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     2401 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     2521 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     2581 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     2641 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     2701 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     2761 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     2821 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     2881 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     2941 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg
     3001 gtacccgggg atccgttaat taa
//
