pGL3-EMX1-U6-Nicking sgRNA

LOCUS      pGL3-EMX1-U6-Nicking sgRNA            4950 bp DNA     circular SYN 12-MAR-2026
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4950)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 4950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
FEATURES             Location/Qualifiers
     source          1..4950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(322..345)
                     /label=OL215
     misc_feature    322..341
                     /label=EMX1 nickingRNA
     primer_bind     322..341
                     /label=OL973
     misc_RNA        342..417
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    469..586
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        635..1145
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1064..1082
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1167..1766
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1167..1186)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1663..1683
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1913..2034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1924..1943)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1978..1997
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2282..2299)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2453..3041)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2533..2552)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3212..4072)
                     /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     3835..3854
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4073..4177)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4204..4659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4291..4310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4501..4522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4790..4838
                     /note="synthetic polyadenylation signal"
     misc_feature    4852..4943
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4892..4911
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gctccccatt ggcctgcttc ggttttagag ctagaaatag
      361 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      421 ttttaaagaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa
      481 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
      541 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
      601 ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag
      661 gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg
      721 gcgccgaccc tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg
      781 ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt
      841 ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc
      901 tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca
      961 atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg
     1021 cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca
     1081 agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc
     1141 cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca
     1201 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     1261 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     1321 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     1381 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     1441 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     1501 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     1561 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     1621 agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg
     1681 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     1741 gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa
     1801 ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag
     1861 atcttagcca ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac
     1921 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1981 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     2041 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     2101 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag
     2161 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     2221 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt
     2281 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2341 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2401 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2461 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2521 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2581 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2641 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2701 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2761 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2821 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2881 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2941 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3001 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3061 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3121 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3181 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3241 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3301 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac
     3361 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3421 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3481 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3541 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3601 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3661 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3721 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3781 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3841 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3901 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3961 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4021 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4081 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4141 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4201 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4261 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4381 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4501 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4561 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4621 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc
     4681 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac
     4741 catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc
     4801 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc
     4861 tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag
     4921 tgcaggtgcc agaacatttc tctatcgata
//