LOCUS       sequence-112660-        2324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The Joung Lab recommends using BPK2660 (Addgene plasmid 70709) 
            instead of VVT1 as guide RNAs cloned into BPK2660 are more effective
            than this plasmid. Human expression plasmid for S. aureus Cas9 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2324)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 2324)
  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: "Nature. 
            2015 Jun 22. doi: 10.1038/nature14592."
COMMENT     Sequence Label: VVT1
FEATURES             Location/Qualifiers
     source          1..2324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(575..1163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(655..674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1334..2125,2126..2194))
                     /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     1957..1976
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2195..2299)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc cgagacgatt aatgcgtctc ggttttagta
      361 ctctgtaatt ttaggtatga ggtagacgaa aattgtactt atacctaaaa ttacagaatc
      421 tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttttaa
      481 gcttgggccg ctcgaggtac ctctctacat atgacatgtg agcaaaaggc cagcaaaagg
      541 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      601 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      661 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      721 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      781 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
      841 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      901 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      961 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1021 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1081 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1141 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1201 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1261 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1321 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1381 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1441 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1501 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1561 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1621 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1681 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1741 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1801 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1861 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1921 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     1981 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2041 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     2101 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2161 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2221 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2281 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acct
//
