LOCUS       sequence-233072-        8066 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 8066)
  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 8066)
  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..8066
                     /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        43..291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     43..63
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     214..233
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        313..388
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        423..802
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1082..2098
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2022..2044
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        2114..2132
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2140..2580
                     /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             2581..2601
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        2657..2675
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2677..2686
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2686..2709
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2880..2896)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2916..3140
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          3361..4299
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4256..4276
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4334..4343
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4340..4342,4343..4345,4346..5059)
                     /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(4385..4406)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    5090..5201
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5319..5336)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5319..5335)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5328..5350)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5476..5931)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5613..5634)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5825..5844
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5958..6062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6063..6131,6132..6923)
                     /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(6281..6300)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7094..7682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7583..7602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7836..7853
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7970..7991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8006..8011,8012..8029,8030..8036)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8044..8060
                     /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(8049..8066,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc ccccccttca ccgagggcct atttcccatg
       61 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      121 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      181 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      241 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgactccagt
      301 ctttctagaa gagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
      361 acttgaaaaa gtggcaccga gtcggtgctt tttttgatcc aagggtgggg cgcgccggat
      421 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      481 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      541 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      601 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      661 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      721 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      781 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      841 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      901 gatgggggcg gggggggggg gggggcgcgc gccrggsggg gsggggsggg gsgrggggsg
      961 gggsggggsg rggsggrgrg gkgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1021 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1081 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1141 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1201 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     1261 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
     1321 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
     1381 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1441 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1501 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1561 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1621 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1681 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1741 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1801 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1861 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1921 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1981 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     2041 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagat
     2101 ccttaattaa taataatacg actcactata ggggccacca tgaagctact gtcttctatc
     2161 gaacaagcat gcgatatttg ccgacttaaa aagctcaagt gctccaaaga aaaaccgaag
     2221 tgcgccaagt gtctgaagaa caactgggag tgtcgctact ctcccaaaac caaaaggtct
     2281 ccgctgacta gggcacatct gacagaagtg gaatcaaggc tagaaagact ggaacagcta
     2341 tttctactga tttttcctcg agaagacctt gacatgattt tgaaaatgga ttctttacag
     2401 gatataaaag cattgttaac aggattattt gtacaagata atgtgaataa agatgccgtc
     2461 acagatagat tggcttcagt ggagactgat atgcctctaa cattgagaca gcatagaata
     2521 agtgcgacat catcatcgga agagagtagt aacaaaggtc aaagacagtt gactgtatcg
     2581 cctaagaaaa agaggaaggt gcacggagga ggtggaagcg gaggaggagg aagcggagga
     2641 ggaggtagcc gattaataat acgactcact ataggggcca ccatggacta caaggatgac
     2701 gacgataaac cctcgacatc cactttgaaa caagttacta aagtggattg tcctgtttgc
     2761 ggggttaaca ttccagaaag tcacattaat aagcatttag acagctgttt atcacgcgaa
     2821 gagaagaagg aaagcctcag aagttctgtt cacaaaaggt agacgcgtaa atgattgcag
     2881 atccactagt tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2941 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3001 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3061 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3121 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgagatccac
     3181 tagttctagc ctcgaggcta gagcggcccc ggtgcctaga gaaggtggcg cggggtaaac
     3241 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3301 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3361 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     3421 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     3481 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     3541 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     3601 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     3661 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     3721 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     3781 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     3841 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     3901 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3961 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     4021 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     4081 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     4141 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     4201 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     4261 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc gatcgcatcg
     4321 acagtgttta aacgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     4381 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg
     4441 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     4501 gcccgtgccc tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg
     4561 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     4621 ccaggagcgc accatcttct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa
     4681 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga
     4741 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaaca tctatatcat
     4801 ggccgtcaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acgtggagga
     4861 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     4921 gctgctgccc gacagccact acctgagcac ccagtccgtg ctgagcaaag accccaacga
     4981 gaagcgcgat cacatggtcc tgctggagtt ccgcaccgcc gccgggatca ctctcggcat
     5041 ggacgagctg tacaagtgag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     5101 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5161 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5221 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     5281 agcaggcatg cgtttaaaca ttagtcgaca gggccgccac tggccgtcgt tttacaacgt
     5341 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5401 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5461 ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5521 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5581 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5641 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5701 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5761 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5821 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5881 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     5941 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6001 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     6061 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6121 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6181 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6241 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6301 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6361 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6421 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6481 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6541 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6601 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6661 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6721 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6781 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6841 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6901 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6961 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7021 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7081 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7141 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7201 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7261 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7321 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7381 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7441 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     7501 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7561 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7621 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7681 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7741 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7801 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7861 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     7921 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7981 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     8041 gaattgtgag cggataacaa tttcac
//
