LOCUS       sequence-119655-        3178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA scaffold with hU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3178)
  AUTHORS   Lonowski LA, Narimatsu Y, Riaz A, Delay CE, Yang Z, Niola F, Duda K,
            Ober EA, Clausen H, Wandall HH, Hansen SH, Bennett EP, Frodin M
  TITLE     Genome editing using FACS enrichment of nuclease-expressing cells 
            and indel detection by amplicon analysis.
  JOURNAL   Nat Protoc. 2017 Mar;12(3):581-603. doi: 10.1038/nprot.2016.165. 
            Epub 2017 Feb 16.
  PUBMED    28207001
REFERENCE   2  (bases 1 to 3178)
  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"; doi: 
            "10.1038/nprot.2016.165"; journalName: "Nat Protoc"; date: 
            "2017-03"; volume: "12"; issue: "3"; pages: "581-603"
COMMENT     Sequence Label: U6GRNA
FEATURES             Location/Qualifiers
     source          1..3178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   7..147
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7..136
                     /label=AAV2 ITR
     rep_origin      222..677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(309..328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     519..540
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(694..713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     813..835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(873..891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        959..1063
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1064..1132,1133..1924)
                     /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(1282..1301)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2095..2683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2584..2603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2745..2993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2745..2765
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2916..2935
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3012..3087
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
       61 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
      121 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
      181 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
      241 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      301 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      361 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      421 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      481 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
      541 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
      601 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
      661 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      721 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      781 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      841 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
      901 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
      961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1021 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1081 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1141 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1201 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1261 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1321 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1381 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1441 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1501 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1561 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1621 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1681 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1741 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     1801 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1861 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     1921 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     1981 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2041 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2101 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2161 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2221 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2281 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2341 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2401 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2461 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2521 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2581 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2641 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2701 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca
     2761 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     2821 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     2881 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     2941 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt
     3001 cgagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     3061 cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa
     3121 aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagag
//
