LOCUS       sequence-238452-        8443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Ncapd3 and Cas9 nickase in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8443)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 8443)
  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 Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: px335-sgRNA-EF(Cas9n)-Ncapd3-R
FEATURES             Location/Qualifiers
     source          1..8443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        452..737
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1016..1243
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1264..1290
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1294..1314
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1324..3039
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5427..5447
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5484..5501)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5490..5697
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5706..5846
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5706..5835
                     /label=AAV2 ITR
     rep_origin      5921..6376
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6008..6027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6218..6239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6393..6412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6512..6534
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6572..6590)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6658..6762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6763..6831,6832..7623)
                     /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(6981..7000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7794..8382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8283..8302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gggccttacg aagggacctc gtttaagagc tatgctggaa acagcatagc
      301 aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      361 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttag cgcgtgcgcc
      421 aattctgcag acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc
      481 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt
      541 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      601 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      661 attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      721 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      781 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      841 gggcgggggg gggggggggc gcgcgccagg cggggcgggg gggggggggg gggggggggg
      901 gggggggggg gggggggsgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
      961 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
     1021 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     1081 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     1141 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
     1201 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
     1261 atgtacccat acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg
     1321 tccgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
     1381 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     1441 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     1501 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     1561 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     1621 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     1681 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     1741 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     1801 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1861 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     1921 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     1981 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
     2041 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     2101 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     2161 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     2221 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     2281 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     2341 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     2401 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     2461 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     2521 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     2581 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     2641 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     2701 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     2761 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     2821 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2881 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     2941 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     3001 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     3061 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     3121 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     3181 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     3241 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     3301 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     3361 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     3421 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     3481 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     3541 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     3601 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     3661 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     3721 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     3781 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     3841 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     3901 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     3961 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     4021 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     4081 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     4141 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     4201 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     4261 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     4321 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     4381 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     4441 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     4501 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     4561 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     4621 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     4681 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     4741 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     4801 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     4861 tttctggaag ccaagggcta caaagaagtg aaaaggacct gatcatcaag ctgcctaagt
     4921 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     4981 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     5041 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     5101 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     5161 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     5221 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     5281 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     5341 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     5401 acctgtctca gctgggaggc gacagcccca agaagaagag aaaggtggag gccagctaag
     5461 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     5521 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5581 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5641 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     5701 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     5761 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     5821 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     5881 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     5941 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     6001 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6061 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6121 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6181 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6241 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     6301 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6361 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6421 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6481 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6541 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     6601 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6661 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6721 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6781 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6841 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6901 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6961 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7021 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7081 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7141 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     7201 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     7261 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     7321 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     7381 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     7441 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     7501 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7561 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7621 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7681 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7921 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8401 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
