LOCUS       sequence-233060-        7672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of fusion protein of yeast Gal4 with human 
            RNF169 Ubiquitin binding domain and BFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7672)
  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 7672)
  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: pCAG-Gal4-RNF169UBD
FEATURES             Location/Qualifiers
     source          1..7672
                     /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"
     enhancer        38..417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          697..1713
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1637..1659
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1729..1747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1755..2195
                     /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             2196..2216
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        2272..2290
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2292..2301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2301..2324
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2486..2502)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2522..2746
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          2967..3905
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3862..3882
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3940..3949
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3946..3948,3949..3951,3952..4665)
                     /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(3991..4012)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4696..4807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4925..4942)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4925..4941)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4934..4956)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5082..5537)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5219..5240)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5431..5450
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5564..5668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5669..5737,5738..6529)
                     /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(5887..5906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6700..7288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7189..7208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7442..7459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7576..7597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7612..7617,7618..7635,7636..7642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7650..7666
                     /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(7655..7672,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc cggattcgac attgattatt gactagttat
       61 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      121 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      181 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      241 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      301 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      361 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      421 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      481 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      541 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggsg grgaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      901 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      961 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1081 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1141 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1201 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1261 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggrgggc
     1321 tcgggggrgg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1381 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1441 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1501 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1561 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1621 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1681 aaccatgttc atgccttctt ctttttccta cagatcctta attaataata atacgactca
     1741 ctataggggc caccatgaag ctactgtctt ctatcgaaca agcatgcgat atttgccgac
     1801 ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc caagtgtctg aagaacaact
     1861 gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct gactagggca catctgacag
     1921 aagtggaatc aaggctagaa agactggaac agctatttct actgattttt cctcgagaag
     1981 accttgacat gattttgaaa atggattctt tacaggatat aaaagcattg ttaacaggat
     2041 tatttgtaca agataatgtg aataaagatg ccgtcacaga tagattggct tcagtggaga
     2101 ctgatatgcc tctaacattg agacagcata gaataagtgc gacatcatca tcggaagaga
     2161 gtagtaacaa aggtcaaaga cagttgactg tatcgcctaa gaaaaagagg aaggtgcacg
     2221 gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagccgatta ataatacgac
     2281 tcactatagg ggccaccatg gactacaagg atgacgacga taaaaagctt cagcaagagg
     2341 aagaagaccg acagttggct ctgcagttgc agcgcatgtt cgacaatgag aggcggactg
     2401 tgagccggcg aaaaggaagt gtggatcagt atctcctacg gtccagcaac atggccgggg
     2461 ccaagtagac gcgtaaatga ttgcagatcc actagttcta gagctcgctg atcagcctcg
     2521 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2581 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2641 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2701 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2761 agaaccagct ggggctcgag atccactagt tctagcctcg aggctagagc ggccccggtg
     2821 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     2881 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     2941 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     3001 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     3061 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     3121 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     3181 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     3241 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     3301 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     3361 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     3421 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     3481 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     3541 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     3601 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     3661 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
     3721 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     3781 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     3841 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     3901 ttcaggtgtc gtgagcgatc gcatcgacag tgtttaaacg ccaccatggt gagcaagggc
     3961 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     4021 cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg
     4081 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     4141 agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     4201 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     4261 acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4321 ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4381 ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat caaggtgaac
     4441 ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4501 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct gagcacccag
     4561 tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttccgc
     4621 accgccgccg ggatcactct cggcatggac gagctgtaca agtgagaatt cctagagctc
     4681 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4741 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4801 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4861 gcaaggggga ggattgggaa gagaatagca ggcatgcgtt taaacattag tcgacagggc
     4921 cgccactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4981 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5041 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
     5101 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5161 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     5221 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5281 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     5341 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     5401 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     5461 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     5521 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5581 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5641 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5701 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5761 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5821 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5881 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5941 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6001 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6061 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6121 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6181 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6241 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6301 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6361 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6421 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6481 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6541 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6601 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6661 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6721 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6781 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6841 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6901 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6961 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7021 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7081 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7141 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7201 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7261 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7321 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7381 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7441 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7501 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7561 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7621 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc ac
//
