pU6-FANCF-PegRNA-H1-Nicking sgRNA

LOCUS      pU6-FANCF-PegRNA-H1-Nicking sgRNA            5308 bp DNA     circular SYN 19-NOV-2025
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  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 5308)
  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..5308
                     /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"
     misc_feature    322..341
                     /label=FANCF spacer
     misc_RNA        342..417
                     /label=5' to 3' scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    418..434
                     /label=RT template
     misc_feature    435..447
                     /label=PBS
     misc_feature    455..670
                     /label=H1 promoter
     primer_bind     458..474
                     /label=OL2772
     primer_bind     590..609
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(662..680)
                     /label=OL2773
     primer_bind     complement(662..680)
                     /label=OL2779
     primer_bind     complement(662..679)
                     /label=OL2776
     misc_feature    680..699
                     /label=FANCF nickingRNA
     misc_RNA        700..775
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    827..944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        993..1503
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1422..1440
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1525..2124
                     /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(1525..1544)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2021..2041
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2271..2392
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2282..2301)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2336..2355
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2640..2657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2811..3399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2891..2910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3570..4430)
                     /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     4193..4212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4431..4535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4562..5017
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4649..4668)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4859..4880
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5148..5196
                     /note="synthetic polyadenylation signal"
     misc_feature    5210..5301
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5250..5269
                     /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 gggaatccct tctgcagcac cgttttagag ctagaaatag
      361 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgga
      421 aaagcgatca aggtgctgca gaagggattt ttttgaacgc tgacgtcatc aacccgctcc
      481 aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag
      541 gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt
      601 tctgggaaat caccataaac gtgaaatgtc tttggatttg ggaatcttat aagttctgta
      661 tgaggaccac gaaacaccgg gggtcccagg tgctgacgtg ttttagagct agaaatagca
      721 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
      781 ttaaagaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      841 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      901 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      961 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
     1021 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     1081 gccgaccctg ggactcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     1141 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     1201 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     1261 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     1321 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     1381 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     1441 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     1501 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     1561 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1621 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1681 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     1741 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1801 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1861 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1921 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1981 cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc gcaacctccc cttctacgag
     2041 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     2101 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     2161 agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     2221 cttagccact ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat
     2281 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2341 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2401 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     2461 gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag
     2521 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2581 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     2641 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2701 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2761 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2821 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2881 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2941 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3001 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3061 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3241 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg ggacccacgc
     3721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4441 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4561 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4621 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4681 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4741 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4801 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4861 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4921 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4981 aacgcgaatt ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc
     5041 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca
     5101 tgataagtaa gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt
     5161 tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc
     5221 tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg
     5281 caggtgccag aacatttctc tatcgata
//