LOCUS       sequence-198391-        8161 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  EBNA episome plasmid for U6 promoter-driven expression of a gRNA 
            targeting EEA-motif. Includes PGK-puro selection cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8161)
  AUTHORS   Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti
            EM, Trokovic R, Kere J, Otonkoski T
  TITLE     Human pluripotent reprogramming with CRISPR activators.
  JOURNAL   Nat Commun. 2018 Jul 6;9(1):2643. doi: 10.1038/s41467-018-05067-x.
  PUBMED    29980666
REFERENCE   2  (bases 1 to 8161)
  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: "Nat 
            Commun."; date: "2018-07-6"; volume: "9(1):2643. doi"; pages: " 
            10.1038/s41467-018-05067-x"
COMMENT     Sequence Label: GG-EBNA-EEA-guide1-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..8161
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    168..201
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      388..2177
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     primer_bind     complement(2746..2765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2865..2887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2925..2943)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3011..3115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3116..3184,3185..3976)
                     /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(3334..3353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4147..4735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4636..4655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5023..5044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5059..5064,5065..5082,5083..5089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5097..5113
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5102..5124
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5121..5137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5156..5175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5156..5174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5257..5274
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5258..5274
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5275..5523
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5275..5295
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5446..5465
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5539..5614
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    5772..5805
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5866..6365
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5901..5924)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6298..6317
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6405..7004
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6405..6424)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 tgtaacttgt taggagacgc cctcaatcgt attaaaagcc gtgtattccc ccgcactaaa
       61 gaataaatcc ccagtagaca tcatgcgtgc tgttggtgta tttctggcca tctgtcttgt
      121 caccattttc gtcctcccaa catggggcaa ttgccggaac ccttaatata acttcgtata
      181 atgtatgcta tacgaagtta ttaggtccct cgaagaggtt cactagcgga tctcaattgg
      241 gcatacccat gttgtcacgt cactcagctc cgcgctcaac accttctcgc gttggaaaac
      301 attagcgaca tttacctggt gagcaatcag acatgcgacg gctttagcct ggcctcctta
      361 aattcaccta agaatgggag caaccagcag gaaaaggaca agcagcgaaa attcacgccc
      421 ccttgggagg tggcggcata tgcaaaggat agcactccca ctctactact gggtatcata
      481 tgctgactgt atatgcatga ggatagcata tgctacccgg atacagatta ggatagcata
      541 tactacccag atatagatta ggatagcata tgctacccag atatagatta ggatagccta
      601 tgctacccag atataaatta ggatagcata tactacccag atatagatta ggatagcata
      661 tgctacccag atatagatta ggatagccta tgctacccag atatagatta ggatagcata
      721 tgctacccag atatagatta ggatagcata tgctatccag atatttgggt agtatatgct
      781 acccagatat aaattaggat agcatatact accctaatct ctattaggat agcatatgct
      841 acccggatac agattaggat agcatatact acccagatat agattaggat agcatatgct
      901 acccagatat agattaggat agcctatgct acccagatat aaattaggat agcatatact
      961 acccagatat agattaggat agcatatgct acccagatat agattaggat agcctatgct
     1021 acccagatat agattaggat agcatatgct atccagatat ttgggtagta tatgctaccc
     1081 atggcaacat tagcccaccg tgctctcagc gacctcgtga atatgaggac caacaaccct
     1141 gtgcttggcg ctcaggcgca agtgtgtgta atttgtcctc cagatcgcag caatcgcgcc
     1201 cctatcttgg cccgcccacc tacttatgca ggtattcccc ggggtgccat tagtggtttt
     1261 gtgggcaagt ggtttgaccg cagtggttag cggggttaca atcagccaag ttattacacc
     1321 cttattttac agtccaaaac cgcagggcgg cgtgtggggg ctgacgcgtg cccccactcc
     1381 acaatttcaa aaaaaagagt ggccacttgt ctttgtttat gggccccatt ggcgtggagc
     1441 cccgtttaat tttcgggggt gttagagaca accagtggag tccgctgctg tcggcgtcca
     1501 ctctctttcc ccttgttaca aatagagtgt aacaacatgg ttcacctgtc ttggtccctg
     1561 cctgggacac atcttaataa ccccagtatc atattgcact aggattatgt gttgcccata
     1621 gccataaatt cgtgtgagat ggacatccag tctttacggc ttgtccccac cccatggatt
     1681 tctattgtta aagatattca gaatgtttca ttcctacact agtatttatt gcccaagggg
     1741 tttgtgaggg ttatattggt gtcatagcac aatgccacca ctgaaccccc cgtccaaatt
     1801 ttattctggg ggcgtcacct gaaaccttgt tttcgagcac ctcacataca ccttactgtt
     1861 cacaactcag cagttattct attagctaaa cgaaggagaa tgaagaagca ggcgaagatt
     1921 caggagagtt cactgcccgc tccttgatct tcagccactg cccttgtgac taaaatggtt
     1981 cactaccctc gtggaatcct gaccccatgt aaataaaacc gtgacagctc atggggtggg
     2041 agatatcgct gttccttagg acccttttac taaccctaat tcgatagcat atgcttcccg
     2101 ttgggtaaca tatgctattg aattagggtt agtctggata gtatatacta ctacccggga
     2161 agcatatgct acccgtttag ggttaacaag ggggccttat aaacactatt gctaatgccc
     2221 tcttgagggt ccgcttatcg gtagctacac aggcccctct gattgacgtt ggtgtagcct
     2281 cccgtagtct tcctgggccc ctgggaggta catgtccccc agcattggtg taagagcttc
     2341 agccaagagt tacacataaa ggcaatgttg tgttgcagtc cacagactgc aaagtctgct
     2401 ccaggatgaa agccactcag tgttggcaaa tgtgcacatc catttataag gatgtcaact
     2461 acagtcagag aacccctttg tgtttggtcc ccccccgtgt cacatgtgga acagggccca
     2521 gttggcaagt tgtaccaacc aactgaaggg attacatgca ctgccccgcg aagaaggggc
     2581 agagatgtcg tagtcaggtt tagttcgtcc ggggcgggga tcgatcctct agtaagggat
     2641 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2701 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     2761 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2821 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2881 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     2941 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3001 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3061 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3301 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4741 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4801 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4861 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4921 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4981 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     5041 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5101 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctctaata
     5161 cgactcacta tagggagaca agcttgcatg cctgcaggtc gactctagag gatctcgagc
     5221 tcaagcttcg aattcggatc ctcgagcgtc tcaccctgta aaacgacggc cagtgagggc
     5281 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     5341 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     5401 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     5461 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     5521 accgcccagc actttggggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     5581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tcatggtcat agctgtttcc
     5641 tcatgtgaga cgagatctgt cgacgcggcc gcaggaaccc actccctctc tgcgcgctcg
     5701 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccaattccga tcatattcaa
     5761 taacccttaa tataacttcg tataatgtat gctatacgaa gttattaggt ctgaagagga
     5821 gtttacgtcc agccaagctt aggatctcga cctcgaaatt ctaccgggta ggggaggcgc
     5881 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     5941 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     6001 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     6061 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     6121 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     6181 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     6241 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     6301 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     6361 tttcgacctg catccatcta gatctcgagc agctgaagct taccatgacc gagtacaagc
     6421 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     6481 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     6541 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     6601 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     6661 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     6721 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     6781 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     6841 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     6901 acaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     6961 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     7021 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gataagctga tcctcacagg
     7081 ccgcacccag cttttcttcc gttgccccag tagcatctct gtctggtgac cttgaagagg
     7141 aagaggaggg gtcccgagaa tccccatccc taccgtccag caaaaagggg gacgaggaat
     7201 ttgaggcctg gcttgaggct caggacgcaa atcttgagga tgttcagcgg gagttttccg
     7261 ggctgcgagt aattggtgat gaggacgagg atggttcgga ggatggggaa ttttcagacc
     7321 tggatctgtc tgacagcgac catgaagggg atgagggtgg gggggctgtt ggagggggca
     7381 ggagtctgca ctccctgtat tcactgagcg tcgtctaata aagatgtcta ttgatctctt
     7441 ttagtgtgaa tcatgtctga cgaggggcca ggtacaggac ctggaaatgg cctaggagag
     7501 aagggagaca catctggacc agaaggctcc ggcggcagtg gacctcaaag aagagggggt
     7561 gataaccatg gacgaggacg gggaagagga cgaggacgag gaggcggaag accaggagcc
     7621 ccgggcggct caggatcagg gccaagacat agagatggtg tccggagacc ccaaaaacgt
     7681 ccaagttgca ttggctgcaa agggacccac ggtggaacag gagcaggagc aggagcggga
     7741 ggggcaggag caggaggggc aggagcagga ggaggggcag gagcaggagg aggggcagga
     7801 ggggcaggag gggcaggagg ggcaggagca ggaggagggg caggagcagg aggaggggca
     7861 ggaggggcag gaggggcagg agcaggagga ggggcaggag caggaggagg ggcaggaggg
     7921 gcaggagcag gaggaggggc aggaggggca ggaggggcag gagcaggagg aggggcagga
     7981 gcaggaggag gggcaggagg ggcaggagca ggaggagggg caggaggggc aggaggggca
     8041 ggagcaggag gaggggcagg agcaggaggg gcaggagggg caggaggggc aggagcagga
     8101 ggggcaggag caggaggagg ggcaggaggg gcaggagggg caggagcagg aggggcagga
     8161 g
//
