LOCUS       sequence-486530-        3196 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of SpyCas9 single guide RNA with Golden 
            Gate-compatible sgRNA spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3196)
  AUTHORS   Shi H, Al-Sayyad N, Wasko KM, Trinidad MI, Doherty EE, Vohra K, 
            Boger RS, Colognori D, Cofsky JC, Skopintsev P, Bryant Z, Doudna JA
  TITLE     Rapid two-step target capture ensures efficient CRISPR-Cas9-guided 
            genome editing.
  JOURNAL   Mol Cell. 2025 May 1;85(9):1730-1742.e9. doi: 
            10.1016/j.molcel.2025.03.024. Epub 2025 Apr 23.
  PUBMED    40273916
REFERENCE   2  (bases 1 to 3196)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.03.024"; journalName: "Mol Cell"; date: 
            "2025-05-1- 1"; volume: "85"; issue: "9"; pages: "1730-1742.e9"
COMMENT     Sequence Label: pKMW493_U6-sgRNA-entry
FEATURES             Location/Qualifiers
     source          1..3196
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   459..599
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   459..588
                     /label=AAV2 ITR
     rep_origin      674..1129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(761..780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     971..992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1146..1165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1265..1287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1325..1343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1411..1515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1516..1584,1585..2376)
                     /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(1734..1753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2547..3135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3036..3055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttggccgcag gaacccctag tgatggagtt
      481 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
      541 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
      601 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
      661 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
      721 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
      781 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      841 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
      901 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      961 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     1021 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     1081 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     1141 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     1201 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     1261 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     1321 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1381 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1441 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1501 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1561 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1621 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1681 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1741 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1801 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1861 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1921 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1981 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2041 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2101 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2161 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2221 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2281 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2341 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2401 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2461 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3181 ccttttgctc acatgt
//
