LOCUS       sequence-232413-        7074 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression of the human RNF169 Ubiquitin binding domain 
            and BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7074)
  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 7074)
  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-RNF169UBD
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          109..1125
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1049..1071
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1138..1157
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1138..1156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1158..1167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1167..1190
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1352..1368)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1388..1612
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          1833..2771
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2728..2748
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2806..2815
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2812..2814,2815..2817,2818..3531)
                     /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(2857..2878)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3562..3673
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3791..3808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3791..3807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3800..3822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3948..4403)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4085..4106)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4297..4316
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4430..4534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4535..4603,4604..5395)
                     /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(4753..4772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5566..6154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6055..6074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6308..6325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6442..6463
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6478..6483,6484..6501,6502..6508)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6516..6532
                     /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     6521..6543
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6540..6556
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6540..6556
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6576..6955
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
       61 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      121 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
      181 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
      241 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
      301 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
      361 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
      421 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
      481 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
      541 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
      601 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
      661 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
      721 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
      781 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
      841 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
      901 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
      961 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     1021 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     1081 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagatcct taattaataa
     1141 tacgactcac tataggggcc accatggact acaaggatga cgacgataaa aagcttcagc
     1201 aagaggaaga agaccgacag ttggctctgc agttgcagcg catgttcgac aatgagaggc
     1261 ggactgtgag ccggcgaaaa ggaagtgtgg atcagtatct cctacggtcc agcaacatgg
     1321 ccggggccaa gtagacgcgt aaatgattgc agatccacta gttctagagc tcgctgatca
     1381 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1441 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1501 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1561 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1621 gcggaaagaa ccagctgggg ctcgagatcc actagttcta gcctcgaggc tagagcggcc
     1681 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     1741 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     1801 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     1861 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     1921 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     1981 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     2041 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     2101 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     2161 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     2221 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     2281 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     2341 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     2401 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     2461 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     2521 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     2581 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     2641 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     2701 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     2761 ttccatttca ggtgtcgtga gcgatcgcat cgacagtgtt taaacgccac catggtgagc
     2821 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2881 aacggccaca agttcagcgt gaggggcgag ggcgagggcg atgccaccaa cggcaagctg
     2941 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3001 accctgagcc acggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     3061 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3121 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3181 atcgagctga agggcgtcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3241 tacaacttca acagccacaa catctatatc atggccgtca agcagaagaa cggcatcaag
     3301 gtgaacttca agatccgcca caacgtggag gacggcagcg tgcagctcgc cgaccactac
     3361 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacagcca ctacctgagc
     3421 acccagtccg tgctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3481 ttccgcaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg agaattccta
     3541 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3601 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3661 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3721 aggacagcaa gggggaggat tgggaagaga atagcaggca tgcgtttaaa cattagtcga
     3781 cagggccgcc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     3841 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     3901 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt
     3961 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4021 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4081 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4141 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4201 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4261 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4321 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4381 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4441 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4501 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4561 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4621 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4681 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4741 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4801 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4861 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4921 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4981 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5041 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5101 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5161 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5221 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5281 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5341 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5701 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     6361 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     6421 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     6481 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     6541 aggaaacagc tatgaccatg aggcgcgccg gattcgacat tgattattga ctagttatta
     6601 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     6661 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     6721 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     6781 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     6841 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     6901 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     6961 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     7021 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg gggg
//
