LOCUS       sequence-391387-        3047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct encoding a S. pyogenes guide RNA targeting the 
            human CLYBL safe harbor locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3047)
  AUTHORS   Wang Q, Liu J, Janssen JM, Goncalves MAFV
  TITLE     Precise homology-directed installation of large genomic edits in 
            human cells with cleaving and nicking high-specificity Cas9 
            variants.
  JOURNAL   Nucleic Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165.
  PUBMED    36928106
REFERENCE   2  (bases 1 to 3047)
  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"; journalName: "Nucleic 
            Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165."
COMMENT     Sequence Label: AY27_pU6.gRNA.CLYBL
FEATURES             Location/Qualifiers
     source          1..3047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        292..540
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     292..312
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     463..482
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        562..637
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(651..668)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(651..667)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(660..682)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(876..895)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     995..1017
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1055..1073)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1141..1245
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1246..1314,1315..2106)
                     /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(1464..1483)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2277..2865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2766..2785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3019..3036
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttcc
      241 atgggatatc gcatgcctgc agagctctag agtccggccg cccccttcac cgagggccta
      301 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      361 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      421 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      481 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      541 gtcctacata ccgttatcct ggttttagag ctagaaatag caagttaaaa taaggctagt
      601 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc actggccgtc
      661 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      721 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      781 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
      841 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
      901 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
      961 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     1021 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     1081 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     1141 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1201 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1261 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1321 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1381 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1441 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1501 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1561 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1621 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1681 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1741 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1801 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1861 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1921 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     1981 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2041 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2101 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2161 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2221 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2281 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2341 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2401 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2461 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2521 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2581 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2641 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2701 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2761 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2821 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2881 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2941 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3001 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaga
//
