LOCUS       sequence-165899-        2866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty sgRNA expression vector. sgRNA sequence can be cloned into 
            its Bbs I site. Its gRNA scaffold has efficient ver. 2 structure..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2866)
  AUTHORS   Fujita T, Yuno M, Fujii H
  TITLE     Allele-specific locus binding and genome editing by CRISPR at the 
            p16INK4a locus.
  JOURNAL   Sci Rep. 2016 Jul 28;6:30485. doi: 10.1038/srep30485.
  PUBMED    27465215
REFERENCE   2  (bases 1 to 2866)
  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: "Sci Rep."; 
            date: "2016-07-28"; volume: "6:30485. doi"; pages: " 
            10.1038/srep30485"
COMMENT     Sequence Label: gRNA Cloning Vector Bbs I ver. 2
FEATURES             Location/Qualifiers
     source          1..2866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        469..717
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     469..489
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     640..659
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(833..857)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1009..1026)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1119..1707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1199..1218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1878..2669,2670..2738))
                     /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     2501..2520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2739..2843)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccacgtg tcttgtccag gcgcgccact cgagaattct gtacaaaaaa gcaggcttta
      421 aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga gggcctattt
      481 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      541 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      601 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      661 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg
      721 tcttcgagaa gacctgttta agagctatgc tggaaacagc atagcaagtt taaataaggc
      781 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct agacccagct
      841 ttcttgtaca aagttggcat tagaattcaa gcttattaat taatggagca caagactggc
      901 ctcatgggcc ttccgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
      961 ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga
     1021 ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa
     1081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1801 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1861 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1921 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1981 ggcttaccat ctggccccag tgctgcaatg ataccgcgag aaccacgctc accggctcca
     2041 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2101 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2161 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2221 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2281 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2341 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2401 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2461 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2521 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2581 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2641 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2701 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2761 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2821 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
