LOCUS       sequence-332828-        3294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpyCas9  nicking sgRNA for EMX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3294)
  AUTHORS   Liu P, Liang SQ, Zheng C, Mintzer E, Zhao YG, Ponnienselvan K, Mir 
            A, Sontheimer EJ, Gao G, Flotte TR, Wolfe SA, Xue W
  TITLE     Improved prime editors enable pathogenic allele correction and 
            cancer modelling in adult mice.
  JOURNAL   Nat Commun. 2021 Apr 9;12(1):2121. doi: 10.1038/s41467-021-22295-w.
  PUBMED    33837189
REFERENCE   2  (bases 1 to 3294)
  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: "Nat 
            Commun."; date: "2021-04-9"; volume: "12(1):2121. doi"; pages: " 
            10.1038/s41467-021-22295-w"
COMMENT     Sequence Label: SpyCas9 EMX1-nicking sgRNA
FEATURES             Location/Qualifiers
     source          1..3294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     652..672
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        654..672
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        719..967
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     719..739
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     890..909
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        989..1064
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1092..1108)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1093..1109)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1133..1152)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1134..1152)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1162..1179)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1162..1178)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1171..1193)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1320..1775
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1407..1426)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1617..1638
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1801..1905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1906..1974,1975..2766)
                     /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(2124..2143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2937..3294,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      661 cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgccc ccttcaccga
      721 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      781 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      841 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      901 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      961 aaacaccgtt gcccacccta gtcattgggt tttagagcta gaaatagcaa gttaaaataa
     1021 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattcgat
     1081 atcaagctta tcgataccgt cgacctcgag ggggggcccg gtacccaatt cgccctatag
     1141 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1201 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1261 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     1321 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1381 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1441 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1501 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1561 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1621 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1681 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1741 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1801 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1861 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1921 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1981 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2041 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2101 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     2161 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2221 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2281 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2341 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2401 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2461 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2521 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2581 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2641 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2701 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2761 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2821 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2881 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2941 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3001 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3061 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3121 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3181 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3241 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacg
//
