LOCUS       sequence-407542-        4656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA-Bsd expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4656)
  AUTHORS   Kato-Inui T, Takahashi G, Ono T, Miyaoka Y
  TITLE     Fusion of histone variants to Cas9 suppresses non-homologous end 
            joining.
  JOURNAL   PLoS One. 2024 May 13;19(5):e0288578. doi: 
            10.1371/journal.pone.0288578. eCollection 2024.
  PUBMED    38739603
REFERENCE   2  (bases 1 to 4656)
  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: "PLoS One.";
            date: "2024-05-13"; volume: "19(5):e0288578. doi"; pages: " 
            10.1371/journal.pone.0288578. eCollection 2024"
COMMENT     Sequence Label: pGB
FEATURES             Location/Qualifiers
     source          1..4656
                     /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"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     complement(1697..1714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1703..1910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1919..2059
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1919..2048
                     /label=AAV2 ITR
     rep_origin      2134..2589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2221..2240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2431..2452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2606..2625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2725..2747
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2785..2803)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2871..2975
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2976..3044,3045..3836)
                     /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(3194..3213)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4007..4595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4496..4515
                     /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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgaaaacctt
     1261 caacatctcc cagcaggatc tagaattagt agaagtagcg acagagaaga ttacaatgct
     1321 ttatgaggat aataaacatc atgtgggagc ggcaattcgt acgaaaacag gagaaatcat
     1381 ttcggcagta catattgaag cgtatatagg acgagtaact gtttgtgcag aagccattgc
     1441 gattggtagt gcagtttcga atggacaaaa ggattttgac acgattgtag ctgttagaca
     1501 cccttattct gacgaagtag atagaagtat tcgagtggta agtccttgtg gtatgtgtag
     1561 ggagttgatt tcagactatg caccagattg ttttgtgtta atagaaatga atggcaagtt
     1621 agtcaaaact acgattgaag aactcattcc actcaaatat acccgaaatt aagaattcct
     1681 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1741 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1801 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1861 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     1921 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     1981 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2041 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2101 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     2161 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     2221 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2281 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2341 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2401 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2461 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     2521 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2581 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     2641 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     2701 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     2761 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2821 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2881 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2941 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3001 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3061 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3121 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3181 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3241 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3301 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3361 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3421 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3481 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3541 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3601 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3661 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     3721 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3781 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3841 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3901 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3961 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4021 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4081 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4141 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4201 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4261 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4321 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4381 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4441 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4501 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4561 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4621 ggttcctggc cttttgctgg ccttttgctc acatgt
//
