LOCUS       sequence-224979-        8062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Rosa26 sgRNA, fusion protein of yeast Gal4 
            with human Rad18 Ubiquitin binding domain and BFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8062)
  AUTHORS   Bashir S, Dang T, Rossius J, Wolf J, Kuhn R
  TITLE     Enhancement of CRISPR-Cas9 induced precise gene editing by targeting
            histone H2A-K15 ubiquitination.
  JOURNAL   BMC Biotechnol. 2020 Oct 23;20(1):57. doi: 
            10.1186/s12896-020-00650-x.
  PUBMED    33097066
REFERENCE   2  (bases 1 to 8062)
  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: "BMC 
            Biotechnol."; date: "2020-10-23"; volume: "20(1):57. doi"; pages: " 
            10.1186/s12896-020-00650-x"
COMMENT     Sequence Label: pU6Rosa-CAG-Gal4-Rad18UBD
FEATURES             Location/Qualifiers
     source          1..8062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..18
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2..18
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        44..292
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     44..64
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     215..234
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        314..389
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        424..803
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        805..1082
                     /label=chicken beta-actin promoter
     intron          1083..2094
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2018..2040
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        2110..2128
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2136..2576
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             2577..2597
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        2653..2671
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2673..2682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2682..2705
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2876..2892)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2912..3136
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          3357..4295
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4252..4272
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4330..4339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4336..4338,4339..4341,4342..5055)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(4381..4402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    5086..5197
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5315..5332)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5315..5331)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5324..5346)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5473..5928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5560..5579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5770..5791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5954..6058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6059..6127,6128..6919)
                     /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(6277..6296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7090..7678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7579..7598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7832..7849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7966..7987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8002..8007,8008..8025,8026..8032)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8040..8056
                     /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     join(8045..8062,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc cccccccttc accgagggcc tatttcccat
       61 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
      121 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      181 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
      241 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgactccag
      301 tctttctaga agagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      361 aacttgaaaa agtggcaccg agtcggtgct ttttttgatc caagggtggg gcgcgccgga
      421 ttcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      481 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      541 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      601 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      661 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      721 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      781 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      841 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      901 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      961 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1021 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1081 cgggagtcgc tgcgtcgcgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1141 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1201 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
     1261 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
     1321 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
     1381 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1441 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1501 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1561 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
     1621 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1681 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
     1741 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1801 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
     1861 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1921 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
     1981 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     2041 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagatcctt
     2101 aattaataat aatacgactc actatagggg ccaccatgaa gctactgtct tctatcgaac
     2161 aagcatgcga tatttgccga cttaaaaagc tcaagtgctc caaagaaaaa ccgaagtgcg
     2221 ccaagtgtct gaagaacaac tgggagtgtc gctactctcc caaaaccaaa aggtctccgc
     2281 tgactagggc acatctgaca gaagtggaat caaggctaga aagactggaa cagctatttc
     2341 tactgatttt tcctcgagaa gaccttgaca tgattttgaa aatggattct ttacaggata
     2401 taaaagcatt gttaacagga ttatttgtac aagataatgt gaataaagat gccgtcacag
     2461 atagattggc ttcagtggag actgatatgc ctctaacatt gagacagcat agaataagtg
     2521 cgacatcatc atcggaagag agtagtaaca aaggtcaaag acagttgact gtatcgccta
     2581 agaaaaagag gaaggtgcac ggaggaggtg gaagcggagg aggaggaagc ggaggaggag
     2641 gtagccgatt aataatacga ctcactatag gggccaccat ggactacaag gatgacgacg
     2701 ataaaccctc gacatccact ttgaaacaag ttactaaagt ggattgtcct gtttgcgggg
     2761 ttaacattcc agaaagtcac attaataagc atttagacag ctgtttatca cgcgaagaga
     2821 agaaggaaag cctcagaagt tctgttcaca aaaggtagac gcgtaaatga ttgcagatcc
     2881 actagttcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2941 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3001 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3061 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3121 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgag atccactagt
     3181 tctagcctcg aggctagagc ggccccggtg cctagagaag gtggcgcggg gtaaactggg
     3241 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3301 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     3361 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     3421 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
     3481 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
     3541 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
     3601 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
     3661 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
     3721 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
     3781 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
     3841 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
     3901 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     3961 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     4021 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     4081 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     4141 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     4201 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     4261 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgagcgatc gcatcgacag
     4321 tgtttaaacg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4381 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgagggg cgagggcgag
     4441 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4501 gtgccctggc ccaccctcgt gaccaccctg agccacggcg tgcagtgctt cgcccgctac
     4561 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4621 gagcgcacca tcttcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc
     4681 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc
     4741 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacatcta tatcatggcc
     4801 gtcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacgt ggaggacggc
     4861 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4921 ctgcccgaca gccactacct gagcacccag tccgtgctga gcaaagaccc caacgagaag
     4981 cgcgatcaca tggtcctgct ggagttccgc accgccgccg ggatcactct cggcatggac
     5041 gagctgtaca agtgagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     5101 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     5161 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     5221 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     5281 ggcatgcgtt taaacattag tcgacagggc cgccactggc cgtcgtttta caacgtcgtg
     5341 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     5401 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     5461 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5521 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5581 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5641 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5701 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5761 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5821 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5881 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     5941 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6001 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     6061 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6121 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6181 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6241 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6301 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6361 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6421 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6481 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6541 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6601 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6661 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6721 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6781 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6841 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6901 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6961 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     7021 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7081 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7141 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7201 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     7261 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7321 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7381 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7441 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7501 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7561 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7621 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7681 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7741 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7801 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     7861 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     7921 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     7981 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     8041 tgtgagcgga taacaatttc ac
//
